Arsenal vice-chairman David Dein has left the club with immediate effect because of "irreconcilable differences" with the rest of the board.
Dein's sudden departure from Arsenal is a stunning development
American billionaire Stan Kroenke has bought 9.9% of Arsenal and more shares from major stakeholder Danny Fiszman.
It sparked takeover talk, but chairman Peter Hill-Wood played down moves.
He revealed remaining board members, who own 45.45% of the remaining shares, had agreed not to dispose of their shares for at least one year.
In a statement, Hill-Wood said: "On behalf of the board I would like to express our gratitude to David Dein for the many years of loyal service he has given to the club.
"We sincerely regret that irreconcilable differences between Mr Dein and the rest of the board have necessitated a parting of the ways."
Earlier in April, ITV plc announced that they had unconditionally sold their 9.99% shareholding interest in Arsenal Holdings plc to American Kroenke's KSE UK INC.
Kroenke Sports Enterprise have also co-owned American Football franchise St Louis Rams following their relocation to Missouri, while NBA side Denver Nuggets and the NHL's Colorado Avalanche are now too under the group's umbrella.
I'm sure Arsene Wenger will be disappointed his friend's leaving
Ex-Gunners boss George Graham
That development was not seen as the first step towards another American takeover of a English club, although the move to buy ITV's stake came less than a month after KSE stated they had "no interest or intention" of buying into Arsenal.
Both Dein, who joined the Arsenal board in 1983, and fellow director Fiszman had previously stated their intention to keep their shares.
Dein, chairman of the G-14 group of Europe's most influential soccer clubs, was instrumental in bringing French coach Arsene Wenger to Highbury in October 1996.
Fellow Premier League clubs Manchester United, Aston Villa and Liverpool are owned by Americans, while champions Chelsea, are owned by Russian billionaire Roman Abramovich.