Stefanishyna had been in her job for nearly a year.
Her departure had been expected for several weeks.
Her departure coincides with a shake-up in recent weeks of top officials, including the replacement of the prime minister and defence minister.
A reshuffle of Ukraine's diplomatic and ambassadorial appointments is also expected.
Stefanishyna, writing on Facebook, said the decision to leave her post was "my own decision, driven by personal circumstances" and said she had accomplished much during her time in the post.
"We boosted the supply of American weapons and maintained support right when the political climate in Washington was changing and not in our favour," she wrote.
Ukraine's Suspilne public broadcaster quoted Stefanishyna last week as telling reporters in Washington DC that Zelenskiy had not sought her resignation.
Stefanishyna said on Facebook that she would address other questions about her circumstances that have arisen in the media.
Ukrainian media outlets said she had informed the president of her intention to leave her post last month.
The reports referred to long-running investigations into alleged irregularities in her asset declaration.
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