Amber Heard said she recalled "rumors" of Depp pushing Moss down a flight of stairs
The Pirates of the Caribbean star, 58, is suing Heard in Virginia for $50 million, saying she defamed him when she said she was a victim of domestic abuse.
Heard, 36, has countersued for $100 million, arguing that Depp smeared her when his lawyer said her claims were a "hoax."
Depp's attorneys on Wednesday called Moss as a rebuttal witness after Heard had testified about a time she feared Depp would push her sister down a staircase.
Heard said she recalled "rumors" of Depp pushing Moss down a flight of stairs and she slapped Depp to stop him from harming her sister.
Moss, who dated Depp from 1994 to 1998, testified that she slipped on a staircase and injured her back during a vacation the couple took in Jamaica.
Depp rushed to help her, carried her to her room and summoned medical services, she said.
"He never pushed me, kicked me or threw me down any stairs," said Moss, who appeared via video from England.
Closing arguments in the case, which is being heard in Virginia, are scheduled for Friday.
Depp in earlier testimony told jurors that he never hit Heard and argued that she was the one who turned violent.
The pair met in 2011 while filming The Rum Diary and wed in February 2015. Their divorce was finalised about two years later.
At the centre of the legal case is a December 2018 opinion piece by Heard that appeared in the Washington Post. The article never mentioned Depp by name, but his lawyer told jurors it was clear that Heard was referring to him when she said she was "a public figure representing domestic abuse."
Depp, once among Hollywood's biggest stars, said Heard's allegations cost him "everything." A new Pirates movie was put on hold, and Depp was replaced in the Fantastic Beasts film franchise, a Harry Potter spinoff.
Heard's attorneys have argued that she told the truth and that her opinion was protected free speech under the U.S. Constitution's First Amendment.
Depp lost a libel case less than two years ago against British tabloid The Sun that labelled him a "wife beater." A London High Court judge ruled that he had repeatedly assaulted Heard.
Depp's lawyers filed the U.S. case in Fairfax County, Virginia, because the Washington Post is printed there. The newspaper is not a defendant.