3,3′-Diindolylmethane (DIM), a major acid-condensation product or metabolite of indole-3-carbinol which is found in cruciferous vegetables, has been shown to have anticancer, anti-inflammatory, and multiple immune stimulating effects. However, its function in bone metabolism is poorly understood. This study evaluated the effect of DIM on bone mass in mice under physiological and pathological conditions. Eight-week-old female mice received injections of a vehicle or 0.1 mg/g of DIM, twice a week for four weeks. We found that DIM treatment significantly increased bone mass as assessed by dual-energy X-ray absorptiometry (DEXA) and micro-computed tomography (μCT). Further, Bone histomorphometric analyses showed that this treatment significantly reduced bone resorption parameters, but did not increase bone formation parameters. Furthermore, we use ovariectomized (OVX)-induced osteoporotic mouse model, and explore function of DIM in skeletal pathological processes. Bone phenotype analyses revealed that the administration of DIM in this study effectively prevented OVX-induced bone loss resulting from increased bone resorption. Our results demonstrated that DIM increased bone mass by suppressing osteoclastic bone resorption in bone metabolism under both physiological and pathological conditions. Accordingly, DIM may be of value in the treatment and the possible prevention of bone diseases characterized by bone loss, such as postmenopausal osteoporosis.