Experimental toxicological studies in laboratory animals and epidemiological human studies have reported a possible association between water fluoridation and osteosarcoma (OSA). To further explore this possibility, a case-control study of individual dogs evaluated by the UC Davis Veterinary Medical Teaching Hospital was conducted using ecologic data on water fluoridation based on the owner's residence. The case group included 161 dogs with OSA diagnosed between 2008-2012. Two cancer control groups included dogs diagnosed with lymphoma (LSA) or hemangiosarcoma (HSA) during the same period (n = 134 and n = 145, respectively). Dogs with OSA were not significantly more likely to live in an area with optimized fluoride in the water than dogs with LSA or HSA. Additional analyses within OSA patients also revealed no significant differences in age, or skeletal distribution of OSA cases relative to fluoride status. Taken together, these analyses do not support the hypothesis that optimal fluoridation of drinking water contributes to naturally occurring OSA in dogs.