'Lean On', released in March, was streamed as of Thursday nearly 528 million times.
It edged out 'Thinking Out Loud', the ballad by English singer-songwriter Ed Sheeran, which just last month was certified with fanfare as the first song streamed more than 500 million times on Spotify.
'Lean On', which features a touch of Indian influence on the hook over a downtempo house beat, was a collaboration between Los Angeles-based electronic group Major Lazer and French producer DJ Snake.
Danish singer MØ sings the chorus, which goes, "Blow a kiss, fire a gun / All we need is somebody to lean on."
MØ, whose real name is Karen Marie Ørsted, wrote on Instagram that she was "speechless and so fucking happy" about the feat. In a subsequent post, she blew a kiss to her fans from a boat headed to Mexico, saying "life doesn't get better".
Major Lazer thanked fans on Facebook, writing, "We only need you to lean on!"
The video for 'Lean On' was shot in India and includes a Bollywood-inspired dance sequence, with Major Lazer member Diplo known for his interest in South Asia.
DJ Snake marked the milestone just as he started a tour of India, as he resumed live-sets following a car accident.
'Lean On' went to number one on the mainstream US singles chart and has also been an especially strong hit in parts of continental Europe, Australia and New Zealand.
Swedish-based Spotify, which says it has 75 million active users, is by far the largest company in the growing industry of streaming, which allows unlimited, on-demand music. As streaming has taken off so recently and rapidly, virtually none of the hits on Spotify's all-time list are more than a few years old.
Other songs in the top five are a remix of Jamaican reggae singer OMI's 'Cheerleader', 'Uptown Funk' by Mark Ronson and Bruno Mars, and 'Take Me to Church' by Hozier -- all released since 2013.
English superstar Adele's new song "Hello" has broken records for its initial performance but is still far below all-time performers with just under 126 million streams on Spotify on Thursday.
In addition to the Spotify record, the success of 'Lean On' also made MØ the highest-placed Danish artist on the influential Billboard Hot 100 list in over 60 years.
The singer was also featured earlier this year as one of Rolling Stone magazine's ‘artists you need to know' and has performed at a string of high-profile international music festivals. She also appeared on Saturday Night Live alongside Iggy Azalea back in October, although that particular performance did not turn out so well.