The 27-year-old secured his first ATP Tour top-10 victory of the season against the former world No.1, who arrived in Florida in peak form after thwarting Carlos Alcaraz's 16-0 start to the season, and finishing as runner-up at Indian Wells.
"I felt good in my first-round match here, and now I feel way better," said Cerundolo.
"I'm recovering that confidence. The level was there, I was just trying to find it. But now, (I am in) the round of 16, so hopefully I can keep it up."
Monday's match began with Cerundolo racing through the opening set, but Medvedev rallied from 3-1 down in the second set to force a decider.
The pair traded high-quality rallies amid the Florida heat with Cerundolo's forehand aggression disrupting Medvedev, while he produced an instinctive winner to end the two-hour, 17-minute encounter.
"I didn't know what to expect ... It was a great match, super tough," Cerundolo added.
"It was my first match against Daniil. He's probably one of the only guys on tour who I have never played. He has had a great year so far, so I didn't expect to be up 6-0 in the first set and a break up in the second."
The defeat ends Medvedev's pursuit of a Tour-leading 20th win of the season, with the 30-year-old exiting with a 19-5 record after already claiming titles in Brisbane and Dubai.
Cerundolo has long thrived in Miami, highlighted by his semi-final run on debut in 2022 before adding quarter-final showings in 2023 and 2025, and will next face Frenchman Ugo Humbert, a 6-4 7-6 (7-2) winner over Kazakh Alexander Shevchenko.
In other results, Canadian eighth seed Felix Auger-Aliassime was ousted 6-3 1-6 6-3 by Terence Atmane of France and American Frances Tiafoe dug deep to see off Czech Jakub Mensik 7-6 (7-4) 4-6 7-6 (13-11).
With AAP.
