The performance marked Bieber's CMA debut and was filmed at California's iconic Hollywood Bowl, which was beautifully lit up in blue and purple.
Bieber was causally dressed in a green beanie, navy t-shirt and dark pants as he sat perched on a stool while belting out the song to an empty venue.
"Whatever these next performers were practicing for 10,000 hours, it's clearly working because this song is one of the most streamed hits in history," co-host, Darius Rucker, said while introducing the performance.
Earlier in the awards ceremony, co-host Reba McIntire joked that it took 10,000 hours to pull off the trio's performance.
“It took us about 10,000 hours to arrange it, but we have Justin Bieber on the CMAs for the first time, with our buddies Dan + Shay,” she teased.
The track was nominated for three awards -- Single of the Year, Musical Event of the Year, and Music Video of the Year -- but did not win any of the categories. Dan + Shay did take home an award for Vocal Duo of the Year.
"10,000 Hours" previously won an iHeartRadio Music Award and a Billboard Music Award.
The music video for the song, which was released last October, features Bieber and his real-life wife, Hailey Bieber, cuddling in bed together. Dan + Shay's wives are likewise featured.
"It's awesome to get to attend their wedding and it was a blast, it was a really good time. And that was like the first place we debuted the song," Smyers told ET last year. "We had never rehearsed it!"
"It was good though, it was really fun and people were out on the dance floor rocking," he added. "It was a good time."
Watch the video below for more on the guys' friendship.