Considered a leader of the Americana genre, Prine released "Tree of Forgiveness" this year, his first collection of new material in 13 years. He owns his own record label in Nashville and mails his records straight to fans.
"I want to thank all of you, all of you that bought the record, and all of you that didn't buy the record," Prine, 71, said. "We'll get you sooner or later."
Isbell, the leading nominee, won for song of the year for "If We Were Vampires," and as a duo/group for his band, the 400 Unit.
He was considered a favorite to win most of the categories he was nominated in thanks to his Grammy-winning record, "The Nashville Sound." In his acceptance speeches, he credited his bandmates and his wife, Amanda Shires, who plays fiddle in the band and sings with him.
Isbell joked that Shires was chastising him for wasting his time watching TV before he went to write "If We Were Vampires," a song about their relationship. "This song almost did not happen for me," he said. "I have to thank my wife Amanda. Beyond being the inspiration for the song as she often is, she was also the motivation for the song."
The band also performed "White Man's World," a song he wrote after Donald Trump's election. Isbell was the sole male nominee going up against Brandi Carlile, Mary Gauthier and Margo Price for album of the year, and also faced Carlile, Price and Lee Ann Womack in the song of the year category.
Despite the wealth of female nominees this year, the only woman to win an award was bluegrass banjo player Molly Tuttle for instrumentalist of the year.
Tyler Childers was named emerging artist of the year, but the Kentucky-bred singer, whose debut album was called "Purgatory," said he preferred to be called a country singer rather than an Americana artist.
"As a man who identifies as country music singer, I feel Americana ... is a distraction of the issues that we are facing on a bigger level as country music singers," Childers said. "It kind of feels like purgatory."
Soul singer Irma Thomas, bluesman Buddy Guy and k.d. lang all received lifetime achievement honors, while while Rosanne Cash received the "Spirit of Americana" Free Speech award.
Cash, daughter of country icon Johnny Cash, earned several standing ovations during her speech and performance in which she called for equal pay for women and stronger gun control.
"I believe that a single child's life is greater, more precious and more deserving of the protection of this nation and the adults in this room than the right to own a personal arsenal of military style weapons," Cash said.
Celebrating the 25-year anniversary of her album "Ingenue" lang talked about her love of country singers like Patsy Cline as she accepted the award on the stage of the famed Mother Church of Country Music, the Ryman Auditorium.
"I am so honored to be in this temple of great music," lang said. "The trailblazers have really left their sweat on this stage."
Thomas sang her classic, "Time Is On My Side," and joked that she felt too young to be receiving a lifetime achievement award. "And at 77, I am only 14," Thomas said.
Guy accepted his lifetime achievement award, while acknowledging the blues is rarely played on radio anymore. But he said while watching the performers during the show, he was inspired.
"If you think you're too old to learn, you better stay at home," Guy said.
Also honored with lifetime achievement honors were Judy Dlugacz and Cris Williamson, founders behind Olivia Records, an all-female record label that started in the 1970s.