Bone loss during mechanical unloading increases fracture risk and is a major concern for the general population and astronauts during spaceflight. The endocannabinoid system (ECS) plays an important role in bone metabolism. One of the main ECS receptors, cannabinoid receptor 1 (CB1), has been studied in regards to basic bone metabolism;​ however, little is known as to how CB1 and the ECS affect bone in different mechanical environments. In this study, we analyzed the influence of global CB1 deficiency and sex on mice during disuse caused by single limb immobilization. Female mice were more sensitive to disuse-induced BV/TV loss than males in both the femoral metaphysis and tibial epiphysis. Genotype also affected bone loss in a sex-dependent manner, with male mice deficient in CB1 receptors (CB1KO) and female wildtype (WT) mice experiencing increased bone loss in both the tibial metaphysis and femoral epiphysis. Genotype affected the response to disuse as CB1KO mice displayed greater changes in femoral ultimate force, along with lower tibial ultimate stress, compared to WT mice. Female mice had a significantly higher femoral, and lower tibial ultimate force compared to male mice. These results reveal that disuse-induced bone loss due to CB1 deficiency is sex-dependent. CB1 deficiency in male mice exacerbated bone loss, while in females CB1 deficiency appeared to protect against disuse-induced bone loss. Regardless of genotype, female mice were more sensitive than males to disuse. These results suggest that CB1 receptors may represent a potential therapeutic target for mitigation of disuse-induced bone loss.