The franchise announced yesterday high performance coach Jon Preston is leaving his role after five years with the organisation.
That comes a day after Glenn Delaney was named as defence coach for the next two seasons.
The departure of Preston means the four main coaches involved in the Highlanders' title win in 2015 have now all departed.
Jamie Joseph and Tony Brown have gone to Japan, while Scott McLeod has joined the All Blacks as defence coach.
Preston (49) first joined the Highlanders in 2013, along with McLeod, as Joseph sought new coaching partners. Brown came on board a year later.
Scrum coach Clarke Dermody, who started in 2014, is still with the Highlanders.
Preston was assistant coach for the side in his first year, but after Brown arrived, moved more into a role of individual player development and skills.
He coached the Highlanders development team and the side at the Brisbane 10s earlier this year and also helped coach the team in its win over the British and Irish Lions in June this year.
Preston, a former All Black inside back, did not return calls yesterday.
Highlanders chief executive Roger Clark said, in a statement, Preston had developed as a coach since his arrival in 2013 and had lifted the skill level of the playing group.
Clark said Preston had decided it was time to look for the next challenge in his career.
Clark said the Highlanders will be looking to replace Preston immediately.
Whether Preston walked after he missed out on the defence job is not known.
With Preston - who had limited coaching experience before he joined the Highlanders - now leaving, the door may open for Otago coach Cory Brown.
Brown said earlier this week he was keen to move up to the next level of Super Rugby and was talking to the franchise about possible roles. Clark said the franchise was keen to have Brown involved, but no role had been agreed.
Brown worked in a similar player development role with the Connacht club in Ireland before returning to New Zealand in 2014.
It could be possible for Brown to carry on the role as Otago head coach in the Mitre 10 Cup and also be involved with the Highlanders, although coaches do not like to be stretched over two teams.
Brown is also employed as the director of rugby for Otago, overseeing coaching right across the province, and it is difficult to see how he could perform this role along with any job with the Highlanders.
The Highlanders for 2018 will be named at the end of next month with the side, minus its All Blacks and Maori All Blacks, set to begin training about the end of November.
In a statement, Preston said his five years with the Highlanders featured many highlights.
''I've learned a lot and enjoyed being part of the Highlanders family. It's been a pleasure to work with so many quality people, see the players' and the team's game grow and to be part of some pretty exciting football.
''For me personally it is important to keep developing and while I will miss the club, I now look forward to future opportunities.''