Despite the rising incidence of osteoporosis (the most common bone disorder) as life expectancy increases worldwide, the genetic and metabolic factors contributing to this multifactorial disease are still poorly understood. This study investigated the role of arginine metabolism in bone formation and its potential for preventing bone loss in postmenopausal osteoporosis. The osteogenic effects of arginine were evaluated in vitro by determining calcium mineral deposition and the expression of marker genes in the human osteoblastic cell line Saos-2. In vivo analyses were conducted in ovariectomized mice treated with arginine, focusing on femoral bone microarchitecture, marker gene expression and serum metabolite profiles. Arginine treatment enhanced calcium deposition and osteoblastic differentiation in vitro. In contrast, however, this treatment had a deleterious effect in vivo, exacerbating trabecular bone loss. These results are particularly relevant given the wide availability of arginine as a dietary supplement, and our findings underscore the necessity of verifying the safety of nutritional supplements in different populations and in the presence of disease.