Legumain is a lysosomal cysteine protease that has been implicated in an increasing amount of physiological and pathophysiological processes. However, the upstream mechanisms regulating the expression and function of legumain are not well understood. Here, we provide in vitro and in vivo data showing that vitamin D₃ (VD₃) enhances legumain expression and function. In turn, legumain alters VD₃ bioavailability, possibly through proteolytic cleavage of vitamin D binding protein (VDBP). Active VD₃ (1,25(OH)₂D₃) increased legumain expression, activity, and secretion in osteogenic cultures of human bone marrow stromal cells. Upregulation of legumain was also observed in vivo, evidenced by increased legumain mRNA in the liver and spleen, as well as increased legumain activity in kidneys from wild-type mice treated with 25(OH)D₃ (50 µg/kg, subcutaneously) for 8 days compared to a control. In addition, the serum level of legumain was also increased. We further showed that active legumain cleaved purified VDBP (55 kDa) in vitro, forming a 45 kDa fragment. In vivo, no VDBP cleavage was found in kidneys or liver from legumain-deficient mice (Lgmn^−/−), whereas VDBP was cleaved in wild-type control mice (Lgmn^+/+). Finally, legumain deficiency resulted in increased plasma levels of 25(OH)D₃ and total VD₃ and altered expression of key renal enzymes involved in VD₃ metabolism (CYP24A1 and CYP27B1). In conclusion, a regulatory interplay between VD₃ and legumain is suggested.