The world number 20, who won the tournament in 2021, took two hours and 36 minutes to get past the four-time Grand Slam winner.
Samsonova's victory sets up a last-16 clash with American world number three Jessica Pegula on Wednesday.
"I was just trying to fight until the end," Samsonova said of her struggles early in the match, adding she wanted "to push and try to find my game."
Osaka broke her opponent twice in the opening set but Samsonova held her serve throughout the second before winning the final five points of a tie-break.
Both exchanged breaks in the final set before the Russian 26-year-old broke a second time, putting her on track for victory.
Check out the match stats here.
Former world number one Osaka's defeat is the latest setback in her comeback since returning from maternity leave last year, having also lost in the first round of the French Open.
Sixth-ranked Madison Keys also failed to make it past the opening round, losing to 2023 Wimbledon winner Marketa Vondrousova 7-5, 7-6 (8/6).
The victory was Vondrousova's first over a top-10 opponent this year as she battles her way back from a shoulder injury.
One of the oldest women's tournaments in the world, the Berlin Open switched from clay to grass courts in 2021 and has since become a popular warm-up event for Wimbledon, which starts at the end of the month.
World number four Zheng Qinwen pulled out of the tournament through injury on Tuesday.
She was replaced by lucky loser Ashlyn Krueger, who later lost to 2022 Wimbledon champion Elena Rybakina of Kazakhstan 6-3, 7-6.
The top four seeds at the tournament, including French Open winner Coco Gauff and runner-up Aryna Sabalenka, along with Pegula and Jasmine Paolini, all enter at the last-16 stage on Wednesday.