Mr Wightman claimed in a hard-hitting resignation statement issued on Friday that he had been threatened with disciplinary action and even expulsion from the party if had voted in favour of an amendment to the Forensic Medical Services (Victims of Sexual Offences Bill) at Holyrood last week.
His departure brought tributes from across the political spectrum and the Greens described it as a “deep disappointment.”
Mr Wightman said: “I understand that the Scottish Green Party has a strong commitment to equalities and trans rights.
"However some of the language, approaches and postures of the party and its spokespeople have been provocative, alienating, confrontational for many women and men.
"It has become evident to me that the sort of open minded public engagement I would like to see take place on this topic is incompatible with a party that has become censorious of any deviation of an agreed line.”
"Put simply, I cannot operate in this kind of environment and Thursday's vote and the discussions that took place around it were the final confirmation of that."
The Greens and the Liberal Democrats opposed an amendment from MSP Johann Lamont to the Forensics Medical legislation last week.
The amendment sought to allow victims of sex crimes to be able to choose the sex, rather than the gender, of the person who examines them afterwards.
Mr Wightman said he had been minded to support the move but faced strong opposition from within his party.
The Lothians MSP said his concerns on the issue have been growing since he was “admonished” for attending a public meeting on the subject in June last year.
"I have been saddened by the intolerance shown by some party members to an open and mature dialogue about the tensions and conflicts around questions of sex and gender in the context of transgender rights and women's rights." he said.
Mr Wightman will continue as an MSP until the current Parliament dissolves in April and said he will continue to work constructively with Green MSPs ahead of next year's Holyrood election.
A spokesman for the party said the Lothians MSP’s decision to quit was a “matter of deep disappointment”.
“Land reform, empowering local democracy and community empowerment are core Green issues, and Andy’s contribution has been very important, but the Greens remain committed to carrying on this agenda without him,” the spokesman said.“The Scottish Greens wish Andy well in whatever he decides to do.”
Mr Wightman has been a member of the Greens for more than ten years. Before entering Parliament he was widely regarded as one of Scotland’s foremost experts on land reform issues, having penned the seminal books Who Own Scotland? and The Poor Had No Lawyers.
Tory MSP Adam Tompkins was among those to pay tribute after the announcement.
“There is no MSP I hold in higher regard than my friend @andywightman,” he said on Twitter.
“We have many, many disagreements but they are always civil and respectful. One of the most informed, free-thinking and intelligent MSPs we have.”