Osaka has returned to the sport this month after two breaks last season to protect her mental health, and had comfortably won her first two matches.
But in 20-year-old Anisimova she came up against a player in red-hot form and, although Osaka did not play badly, she was beaten in a deciding first-to-10-points tie-break, losing 4-6 6-3 7-6 (5).
The result will be particularly disappointing because Osaka held two match points at 4-5 in the deciding set, but Anisimova saved them both and will go forward to face world number one Ashleigh Barty.
A prodigious junior talent, Anisimova reached the French Open semi-finals as a 17-year-old but had struggled since the death of her father and coach Konstantin later in 2019.
She is ranked only 60th in the world but is unbeaten in 2022 having won the warm-up title in Melbourne and showed here that she remains one of the brightest prospects in the game.
Anisimova is cut from the same cloth as Osaka in terms of the crispness and power of her ball-striking and this was a heavyweight contest from the start.
Osaka played a calm and composed first set but Anisimova cut down on her errors in the second to level the match and there was virtually nothing to choose between the two in a high-quality decider.
Anisimova edged in front early in the tie-break, though, and was almost flawless, dropping her racket to the court in disbelief after clinching victory with an ace on her first match point.
“Going into this match I knew I had to be playing sharp,” said Anisimova. “Naomi is an absolute champion so I knew I had to really step up my game. I think I started doing that in the second set. I’m so grateful I was able to play so well today and get this win. It means a lot.”
A potential meeting between Osaka and Barty had been highlighted as soon as the draw was made but now it is the Australian hero who will have to try to stop Anisimova.
Barty has been untroubled so far and defeated big-hitting Italian Camila Giorgi 6-2 6-3.
Two-time former champion Victoria Azarenka has also breezed through the draw so far, dropping just nine games in three matches.
The 2012 and 2013 champion is having her best run in Melbourne for six years and was in excellent form in beating long-time top-10 star Elina Svitolina 6-0 6-2.
Azarenka next faces fourth seed Barbora Krejcikova, who recovered from a set and a break down to defeat another French Open champion in Jelena Ostapenko 2-6 6-4 6-4.
Krejcikova was unhappy to receive a warning for taking too long over a bathroom break – a rule introduced following the furore at the US Open that surrounded Stefanos Tsitsipas’ lengthy departures from the court.
Another very tight contest saw eighth seed Paula Badosa defeat young Ukrainian Marta Kostyuk 6-2 5-7 6-4.
Badosa next faces a resurgent Madison Keys, who won the WTA Tour title in Adelaide last weekend and moved through to the last 16 by winning a deciding tie-break against Wang Qiang.
Fifth seed Maria Sakkari eased to a 6-4 6-1 victory over Veronika Kudermetova and will face Jessica Pegula next.