Attorney John Dowd confirmed his decision in an email, saying “I love the President and wish him well”.
Mr Dowd’s departure comes three days after the Trump legal team added a new lawyer, former US attorney Joseph diGenova.
The resignation came as Mr Trump furthered his feud with former vice-president Joe Biden.
The American president bizarrely claimed his rival would go “down hard and fast” in any fist fight.
Mr Trump tweeted: “Crazy Joe Biden is trying to act like a tough guy. Actually, he is weak, both mentally and physically, and yet he threatens me, for the second time, with physical assault.
“He doesn’t know me, but he would go down fast and hard, crying all the way. Don’t threaten people Joe!”
Mr Biden – a potential 2020 election rival – had earlier suggested he would “beat the hell out of” Mr Trump if they were in high school.
Mr DiGenova has alleged on television FBI officials were involved in a “brazen plot” to exonerate Hillary Clinton in the email investigation and “frame” Mr Trump for nonexistent crimes.
It already was a delicate time in special counsel Robert Mueller’s investigation.
Mr Trump’s attorneys have been negotiating with Mr Mueller over the scope and terms of an interview of the president.
Mr Trump has told reporters he was eager to speak with Mr Mueller, but Mr Dowd had been far more apprehensive.
The lawyers have not publicly committed to making the president available for questioning.
Over the weekend, Mr Dowd issued a statement calling for an end to special counsel’s investigation. The White House and later Mr Dowd had to clarify the statement, saying the president’s legal team wasn’t calling for Mr Mueller to be fired.
Another Trump lawyer, Jay Sekulow, also confirmed Mr Dowd’s resignation.
“John Dowd is a friend and has been a valuable member of our legal team,” he said.
“We will continue our ongoing representation of the president and our co-operation with the Office of Special Counsel.”
This is at least the second major reshuffling of Mr Trump’s legal team in the past year.
Mr Dowd had taken over the lead lawyer role last summer from New York attorney Marc Kasowitz, who has long been by Mr Trump’s side.