Background and PurposeChronic pain is often a symptom of knee osteoarthritis (OA) for which current analgesics are either inadequate or are associated with serious side effects. The endocannabinoid system may offer alternative targets for pain relief. We evaluated the effects of a potent and selective monoacylglycerol (MAG) lipase inhibitor (MJN110) on OA pain behaviour, spinal mechanisms of action and joint histopathology in the rat.Experimental ApproachIntra‐articular injection of monosodium iodoacetate (MIA) models OA pain and mimics clinical joint pathology. Effects of MJN110 on MIA‐induced weight‐bearing asymmetry and lowered paw withdrawal thresholds (PWTs), changes in spinal gene expression and brain levels of relevant lipids were determined.Key ResultsAcute MJN110 (5 mg·kg−1) significantly reversed MIA‐induced weight‐bearing asymmetry (MIA/vehicle: 68 ± 6 g; MIA/MJN110: 35 ± 4 g) and lowered ipsilateral PWTs (MIA/vehicle: 7 ± 0.8 g; MIA/MJN110: 11 ± 0.6 g), via both CB1 and CB2 receptors. Repeated treatment with MJN110 (5 mg·kg−1) resulted in anti‐nociceptive tolerance. A lower dose of MJN110 (1 mg·kg−1) acutely inhibited pain behaviour, which was maintained for 1 week of repeated administration but had no effect on joint histology. MJN110 significantly inhibited expression of membrane‐associated PGE synthase‐1 in the ipsilateral dorsal horn of the spinal cord of MIA rats, compared with vehicle‐treated MIA rats. Both doses of MJN110 significantly elevated brain levels of the endocannabinoid 2‐arachidonoylglycerol.Conclusions and ImplicationsOur data support further assessment of the therapeutic potential of MAG lipase inhibitors for the treatment of OA pain.