Emergency services attended but the girl was pronounced dead at the scene.
Police said enquiries are ongoing to establish the full circumstances behind the fatal crash.
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The A76, which passes through New Cumnock, was closed for several hours for investigation work.
A Police Scotland spokesperson said: “Around 12.35pm on Monday, 18 September, 2023, we received a report of a crash involving a tractor towing a trailer and a 9-year-old female, on her bicycle on Castle Place, New Cumnock.
“Emergency services attended but the girl was pronounced dead at the scene.
“Enquiries are ongoing to establish the full circumstances.”