Space One, hoping to become the first private Japanese firm to put a satellite in space, said that what went wrong was "under investigation".
TV footage showed the Kairos rocket blasting off from the launch site in the coastal Wakayama region of western Japan at 11:10 am (0210 GMT) up into the blue sky.
But shortly afterwards, the slim, white 18-metre (60-foot) rocket appeared to run out of power and head back downwards in a spin.
It was unclear where it landed.
Tokyo-based Space One told a press conference that "no major anomalies" had been identified within the aircraft itself and the rocket had not deviated from its flight path.
"So it seems reasonable to assume that some issue occurred within the flight interruption system," the firm said, referring to a safety mechanism capable of automatically ending the flight.
The rocket was carrying several satellites including one for a high school in Tokyo, according to the firm.
The company's first launch attempt ended in spectacular failure in March 2024 when the solid-fuel rocket exploded seconds after lift-off.
A second try started off better only for the rocket to suddenly spiral downwards after engineers terminated the operation because of a technical problem.
The latest launch had been slated to happen on Wednesday, only to be cancelled due to another glitch just 28.9 seconds before lift-off.
- Moon landing -
Companies such as Space One want to offer cheaper and more frequent space exploration opportunities than governments.
The startup is hoping to establish a satellite-launching service to tap into expanding global demand -- emulating Elon Musk's SpaceX, which has contracts with NASA and the Pentagon.
Space One was founded in 2018 by businesses including Canon Electronics, IHI Aerospace, construction firm Shimizu and the government-run Development Bank of Japan.
The national Japan Aerospace Exploration Agency (JAXA) is also on a mission to become a major player for satellite launches.
JAXA's next-generation H3 launch system had experienced multiple failed take-off attempts before a successful blast-off in February 2024.
In December, however, Japan's flagship H3 rocket failed to launch a geolocation satellite into orbit due to engine failure.
In 2024, JAXA landed an unmanned probe on the Moon -- albeit at a crooked angle -- making it just the fifth country to achieve a "soft landing".
The Smart Lander for Investigating Moon (SLIM), dubbed the "Moon Sniper" for its landing precision, nonetheless intermittently sent back signals for many months.
And last year Japan's ispace failed in its attempt to become only the third private firm -- and the first outside the United States -- to achieve a controlled arrival on the Moon.
Contact with the ispace's unmanned Resilience craft was lost as the probe made its final descent onto the lunar surface and was believed to have crash landed.
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