5-Lipoxygenase catalyzes leukotriene generation from arachidonic acid. The gene that encodes 5-lipoxygenase, Alox5, has been identified in genome-wide association and mouse Quantitative Trait Locus studies as a candidate gene for obesity and low bone mass. Thus, we tested the hypothesis that Alox5(-/-) mice would exhibit metabolic and skeletal changes when challenged by a high-fat diet (HFD). On a regular diet, Alox5(-/-) mice did not differ in total body weight, percent fat mass, or bone mineral density compared with wild-type (WT) controls (P < 0.05). However, when placed on a HFD, Alox5(-/-) gained more fat mass and lost greater areal bone mass vs. WT (P < 0.05). Microarchitectural analyses revealed that on a HFD, WT showed increases in cortical area (P < 0.01) and trabecular thickness (P < 0.01), whereas Alox5(-/-) showed no change in cortical parameters but a decrease in trabecular number (P < 0.05) and bone volume fraction compared with WT controls (P < 0.05). By histomorphometry, a HFD did not change bone formation rates of either strain but produced an increase in osteoclast number per bone perimeter in Alox5(-/-) mice (P < 0.03). In vitro, osteoclastogenesis of marrow stromal cells was enhanced in mutant but not WT mice fed a HFD. Gene expression for Rankl, Pparg, and Cox-2 was greater in the femur of Alox5(-/-) than WT mice on a HFD (P < 0.01), but these increases were suppressed in the Alox5(-/-) mice after 8 wk of treatment with celecoxib, a cyclooxygenase-2 inhibitor. In sum, there is a strong gene by environmental interaction for bone mass when mice lacking the Alox5 gene are fed a HFD.