Earning the first standing ovation of the night, the trio strolled onto the stage of the 69th annual Emmy Awards to the film’s theme song.
As the performers took their positions at the podium, Parton said she had been waiting for the cast to re-unite since the film was first released.
Fonda told the crowd: “Back in 1980 in that movie, we refused to be controlled by a sexist, egotistical, lying, hypocritical bigot.”
“And in 2017, we still refuse to be controlled by a sexist, egotistical, lying, hypocritical bigot,” Tomlin replied.
Parton did not speak during the exchange but raised her eyebrows, but later got a laugh from the crowd when she said she was hoping to get a "Grace & Frankie" vibrator in her swag bag, referencing Tomlin and Fonda’s Netflix show.
After the appearance, the official Twitter account for "Grace & Frankie" tweeted a GIF of a vibrator to Parton, saying: “We got you”.
The evening was filled with jokes about the president. Host Steven Colbert suggested Mr Trump may not have run for office if he had been awarded an Emmy for The Apprentice, while Alec Baldwin, who won an Emmy for Outstanding Supporting Actor in a Comedy Series for his portrayal of Trump on Saturday Night Live, said: “I suppose I should say, at long last, Mr President, here is your Emmy,” as he accepted the award.
Julia Louis-Dreyfus, who won her sixth consecutive award for Best Actress in a Comedy Role for Veep, joked on stage that they had planned a storyline about impeachment “but we abandoned that because we were worried someone else might get to it first”.
Additional reporting by AP