Kyabram police Sergeant Dale Sim said a B-double truck carrying 63 cattle failed to take a bend on the Murray Valley Hwy at Wyuna about 3pm.
The driver lost control of the truck which had been travelling south on the highway, and the truck tipped over near Pell Rd.
The man driving the truck was not injured in the crash.
Twenty-two cattle, however, either died in the crash or had to be euthanised by vets for animal welfare reasons.
The section of the highway between Bunyip Rd and Agnes Rd was also closed for about four hours, with traffic diverted while the site was cleared.
Police, and staff from Agriculture Victoria, VicRoads, SES, CFA, and Campaspe Shire rangers attended the scene.
Sgt Sim said police investigations into the crash were continuing.
Anyone with information about it is asked to contact Campaspe highway patrol police on 5483 1500 or Crime Stoppers on 1800 333 000 or crimestoppersvic.com.au
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