Playing in the renamed Otago Stadium, the West Africans banged in two second half goals within a few minutes to shock Mexico, a pre tournament favourite.
The result ensured the so called ''group of death'' lived up to its name, and means Mexico has it all to do in its final two group matches, against Uruguay and Serbia.
Adama Traore headed in the first goal in the 76th minute, via a pinpoint Youssouf Kone free kick, before Dieudonne Gbakle sealed the win with his left boot three minutes later.
Gbakle, the standout Mali player throughout the match, added the second when he danced through a few defenders into the box, before sliding the ball past goalkeeper Raul Gudino and into the bottom right hand corner.
The goals followed a flurry of second half red cards, two to Mexico and one to Mali.
Mexico lost forward Diego Gama, a second half substitute, and Oscar Bernal in the 66th and 69th minutes, while Mali lost defender Hamidou Maiga in the 56th.
Maiga received his marching orders after picking up two yellow cards within five minutes. He fouled Gama and Hirving Lozano in the 51st and 56th minutes, leaving German referee Felix Zwayer no choice but to send him packing.
But just when Mexico thought it had an advantage, it was reduced to nine men. Gama dragged down Diadie Samassekou by his collar, a needless foul off the ball.
Zwayer did not hesitate in going straight for the red card.
Just a few moments later, he was reaching into his pocket again, this time for a second yellow card for Bernal, who had been booked early in the first half.
Mexico's Alejandro Diaz should have opened the scoring late in the first half, but his wide open header was somehow parried over the goal by goalkeeper Djigui Diarra.
Erick Aguirre created the chance with a lovely run down the right hand flank, before delivering the cross for Diaz. While Mali's marking inside the box left a lot to be desired, Diarra did brilliantly to knock Diaz's header over.
Mexico looked the more threatening in the opening 45 minutes, particularly winger Hirving Lozano, the team's top scorer during qualification.
He caused all sorts of problems down the right hand side with his pace and quick feet, testing Mali out with a couple of probing crosses inside the first two minutes of the match.
Perhaps Mali, which arrived in Dunedin on Wednesday morning, benefited from the relaxed build up to the game.
Coach Fanyeri Diarra said there was no pressure on his team during the week, and he just wanted his players to enjoy the atmosphere. That is precisely what they did, and now they have taken a giant step towards the round of 16.
Mexico v Mali
The scores
Mali 2
Adama Traore, 56, Dieudonne Gbakle, 59.
Mexico 0
Halftime: 0-0
Crowd: More than 6000
In summary
Mali made sure group D got off to the most improbable start with a 2-0 win against one of the pre tournament favourites.
Mexico will rue a three minute period early in the second half, when it had two players red carded.
Star player
Dieudonne Gbakle sealed the win with a classy finish off his left boot and used his pace and nimble feet to catch Mexico out on many occasions.
Hamilton games
Colombia 1 (Joao Rodriguez) 1, Qatar 0. Halftime: 1-0.
Portugal 3 (Gelson Martins, Nuno Santos, Andrew Silva), Senegal 0. Halftime: 1-0.
