Authorities said they were investigating a white supremacist as the suspect. The assailant was hospitalized in critical condition, Washington, D.C., Mayor Adrian Fenty said.
D.C. Police Chief Cathy Lanier said the gunman was "engaged by security guards immediately after entering the door" with a rifle. "The second he stepped into the building he began firing."
One law enforcement official said James Von Brunn, 89, a white supremacist, was under investigation in the shooting, and a second official said the elderly man's car was found near the museum and tested for explosives. They spoke on condition of anonymity, saying they were not authorized to discuss the investigation just beginning.
Von Brunn has a racist, anti-Semitic website and wrote a book titled "Kill the Best Gentiles."
In 1983, he was convicted of attempting to kidnap members of the Federal Reserve Board. He was arrested two years earlier outside the room where the board was meeting, carrying a revolver, knife and sawed-off shotgun. At the time, police said Von Brunn wanted to take the members hostage because of high interest rates and the nation's economic difficulties.
Writings attributed to Von Brunn on the Internet say the Holocaust was a hoax and decry a Jewish conspiracy to "destroy the white gene pool."
"At Auschwitz the 'Holocaust' myth became Reality, and Germany, cultural gem of the West, became a pariah among world nations," the writing said.
The museum houses exhibits and records relating to the Holocaust more than a half century ago in which more than six million Jews were killed by the Nazis.
Museum officials identified the guard who was killed as Stephen T. Johns. In an e-mail, director Sara Bloomfield said he "died heroically in the line of duty."
The museum where he worked is located across the street from the National Mall, and within sight of the Washington Monument. The museum, which draws about 1.7 million visitors each year, was closed for the day after the shooting, and nearby streets were cordoned off by police. Surrounding roads were closed at least temporarily and blocked off with yellow tape. Police cars and officers on horseback surrounded the area.
The attack was the third in a recent wave of unsettling shootings that appeared to have political underpinnings.
A 23-year-old Army private, William Andrew Long, was shot and killed outside a recruiting office this month in Arkansas and a fellow soldier was wounded. The suspect, a Muslim convert, has said he considers the killing justified because of the U.S. military presence in the Middle East.
Late last month, abortion provider Dr. George Tiller was shot to death in his church.
At the White House a few blocks away from the Museum, press secretary Robert Gibbs said he informed President Barack Obama of the events and the chief executive was "obviously saddened by what has happened."
Only last week, Obama visited the site of a German concentration camp at Buchenwald in Germany where he noted, "There are those who insist the Holocaust never happened." He added, "This place is the ultimate rebuke to such thoughts, a reminder of our duty to confront those who would tell lies about our history."
The museum normally has a heavy security presence with guards positioned both inside and outside. All visitors are required to pass through metal detectors at the entrance, and bags are screened.
Linda Elston, who was visiting the museum from Nevada City, Calif., said she was on the lower level of the museum watching a film when she and others were told to evacuate.
"It was totally full of people," Elston said. "It took us a while to get out."
She said she didn't hear any shots and didn't immediately know why there was an evacuation. The experience left her feeling "a little anxious," she said.
A museum official said a couple of thousand people were inside the facility when the shooting broke out.
The museum, the largest U.S. memorial to the Holocaust, opened in 1993 and has been a target of at least one domestic terrorism threat in the past. In 2002, prosecutors said two members of white supremacist groups had plotted to build a fertilizer bomb - like the one used to destroy an Oklahoma City federal building - to blow up the Holocaust museum. Authorities said the two had plotted to incite a race war.