"Glory to Hong Kong" was created and popularised during the city's widespread 2019 pro-democracy protests, and has in recent months been accidentally played at international sporting events as the city's anthem, drawing the government's ire.
After Beijing imposed a draconian national security law in Hong Kong to quell dissent in 2020, authorities said the song promoted separatist ideas and banned it from schools.
The government last week took the further step of asking Hong Kong's courts to ban the song, with judges scheduled to hear the injunction application next month.
The song was no longer available on streaming platforms Spotify or iTunes as of Friday, though several versions were still available on YouTube.
For months, Hong Kong officials have criticised search giant Google for including "Glory to Hong Kong" in its search results, which they say has contributed to the mix-ups at sporting events held abroad.
Since last year, organisers at several events have mistakenly played the protest song as Hong Kong's anthem, instead of using the Chinese national anthem "March of the Volunteers".
Google previously said it did not manipulate search results and refused to delist the song.
After the anthem was removed from Spotify on Wednesday, its anonymous creator DGX Music said in a Facebook post that it was handling "technical issues unrelated to streaming platforms" which led to "temporary impact".
Spotify told local media that the song was removed by its distributors and not by the platform.
Last week, the song topped download charts on iTunes for days, after the government filed the injunction order.
Apple did not respond to a request for comment.
Under the security law, Hong Kong police can order internet platforms to remove information deemed to be "endangering national security".
Failure or refusal to comply can lead to six months in jail and a maximum fine of HK$100,000 ($12,800).
hol/aha/sco
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