Osteoarthritis (OA) is a degenerative joint disease that causes pain and bone deterioration driven by an increase in prostaglandins and inflammatory cytokines. Current treatments focus on inhibiting prostaglandin production, a pro-inflammatory lipid metabolite, with NSAID drugs; however, other lipid signaling targets could provide safer and more effective treatment strategies. Epoxides of polyunsaturated fatty acids are anti-inflammatory lipid mediators that are rapidly metabolized by the soluble epoxide hydrolase (sEH) into corresponding vicinal diols. sEH inhibitors (sEHI) stabilize these biologically active, anti-inflammatory lipid epoxides and are analgesic in both neuropathic and inflammatory pain conditions. Additionally, increased diols have been observed in the synovial fluid of humans with OA, warranting further research on the biological role of this pathway in the progression of OA. Most experimental studies testing the analgesic effects of sEH inhibitors have used experimental rodent models which do not completely represent the complex etiology of painful diseases. Here, we tested the efficacy of sEH inhibitors in aged dogs with natural arthritis to provide a better representation of the clinical manifestations of pain. Two sEHI were administered orally, once daily for five days to dogs with naturally occurring arthritis to assess efficacy and pharmacokinetics. Blinded technicians recorded the behavior of the arthritic dogs based on pre-determined criteria to assess pain and function. After five days, EC1728 significantly reduced cumulative pain at a dose of 5 mg/kg compared to vehicle controls. Pharmacokinetic evaluation showed concentrations exceeding the enzyme potency in both plasma and synovial fluid. In vitro data showed that epoxyeicosatrienoic acid (EETs), epoxide metabolites of arachidonic acid, decreased inflammatory cytokines, IL-6 and TNF-α, in canine chondrocytes challenged with IL1β to simulate an arthritic environment. These results provide proof of concept that inhibiting the sEH is a non-NSAID, non-opioid strategy for treating osteoarthritis.