Temporomandibular disorders (TMD) are complex pathologies responsible for chronic orofacial pain. Intramuscular injection of botulinum toxin A (BoNT/A) has shown effectiveness in knee and shoulder osteoarthritis, as well as in some TMDs such as masticatory myofascial pain, but its use remains controversial. This study aimed to evaluate the effect of intra-articular BoNT/A injection in an animal model of temporomandibular joint osteoarthritis. A rat model of temporomandibular osteoarthritis was used to compare the effects of intra-articular injection of BoNT/A, placebo (saline), and hyaluronic acid (HA). Efficacy was compared by pain assessment (head withdrawal test), histological analysis, and imaging performed in each group at different time points until day 30. Compared with the rats receiving placebo, those receiving intra-articular BoNT/A and HA had a significant decrease in pain at day 14. The analgesic effect of BoNT/A was evident as early as day 7, and lasted until day 21. Histological and radiographic analyses showed decrease in joint inflammation in the BoNT/A and HA groups. The osteoarthritis histological score at day 30 was significantly lower in the BoNT/A group than in the other two groups ( = 0.016). Intra-articular injection of BoNT/A appeared to reduce pain and inflammation in experimentally induced temporomandibular osteoarthritis in rats.