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Police were called to the A31 at Holybourne near Alton after reports that six horses had escaped from a nearby field.
The incident happened shortly after 7pm when two of the horses, which were being corralled into a trailer, bolted into the road and were in collision with a Toyota Previa. The animals were too severely injured to be treated and were put down at the scene by a vet.
The driver of the vehicle, a 41-year-old woman from Farnham, Surrey and four passengers, a seven-year-old boy, two 10-year-old boys and a four-year-old girl, were shaken but uninjured.
The remaining four horses also escaped and ran along the southbound carriageway to London Road in Holybourne, where they were rounded up in the nearby theatre car park and returned to a secured field by agents of the owners.
The road was re-opened shortly after 9pm.
Any witnesses to the incident are asked to contact PC Phillipa Churcher-Brown on 101.