Osaka, who won her first major title in Indian Wells last weekend, was superior throughout and fully deserved her 6-3, 6-2 win in just one hour and 17 minutes.
It was Williams’ fourth match and second tournament appearance since returning from 13 months maternity leave, yet the American’s movement and inability to stay in the rallies suggested her comeback could be more arduous than expected.
The defeat was the first time Williams has lost her first match at an American hard court championship for 21 years.
Williams, 36, may have been unfairly unseeded here due to her lack of action over the past year could have no complaints with the defeat.
In her comeback in Indian Wells, she made it through to round three where she fell to sister Venus in straight sets.
Taking on Osaka was always going to prove a tough task – and so it proved.
Williams, an eight-time champion in Key Biscayne, started strongly and held her serve to love but it took a while for the American to find her range.
Osaka, 20, showed some understandable nerves but was able to stay in the game and when she broke in the seventh game, it was the perfect chance for the Japanese player to take control.
The world number 22’s serve – which was being clocked at an impressive 115 mph – as well as the power in her ground strokes allowed her to stretch the lead to 5-3.
Osaka was hitting the ball beautifully and broke again to take the first set with relative ease.
Williams’ effort couldn’t be faulted though it was Osaka who possessed the greater firepower and consistency which allowed her to dominate most of the exchanges.
She broke once more to make it 3-1 in the second and the excellent Japanese never looked back, sealing the win when Williams hit an easy passing shot long.
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