Objectives <p>To investigate the role of 5-lipoxygenase (5-Lox) in driving joint inflammation and damage in a model of osteoarthritis induced by collagenase in mice.</p> Methods <p>An osteoarthritis model was established in adult SV129 wild-type (WT) and 5-Lox-deficient (5-Lox^−/−) mice by intra-articular injection of collagenase VII into the tibiofemoral cavity on days 1 and 3. A group of WT mice injected with collagenase was treated with zileuton, an inhibitor of 5-Lox. At different time points after collagenase injection, the expression of 5-Lox and metalloproteinases was assessed using immunohistochemistry and PCR, respectively. Histopathological markers of tissue inflammation and damage were analyzed in whole tissue by HE and safranin O/fast green staining.</p> Results <p>The injection of collagenase increased the protein expression of 5-Lox at 7–21&#xa0;days after this challenge, returning to the basal level on day 42. The expression of Lta4-hydrolase, a critical enzyme involved in leukotriene B4 production, also increased with time after collagenase injection. Compared with WT mice, 5-Lox^−/− mice presented less inflammation, including reduced cell infiltration and synovial hyperplasia, at 42&#xa0;days after collagenase insult. Furthermore, 5-Lox^−/− mice also presented reduced cartilage degradation and lower expression of metalloproteinases 3 and 13 in affected joints. Interestingly, all the inflammatory parameters and degree of cartilage damage improved in the mice treated with zileuton when the compound was given 21&#xa0;days after collagenase injection.</p> Conclusion <p>5-Lox is critical for joint inflammation and degradation in a mouse model of osteoarthritis. Inhibiting 5-Lox with Zileuton effectively prevented joint damage and inflammation, even when it was given weeks after the inciting stimulus, suggesting that it is a potential strategy to limit the progression of osteoarthritis.</p>