Systemic iron homeostasis is maintained by regulation of iron absorption in the duodenum, iron recycling from erythrocytes, and iron mobilization from the liver and is controlled by the hepatic hormone hepcidin. Hepcidin expression is induced via the bone morphogenetic protein (BMP) signaling pathway that preferentially uses two type I (ALK2 and ALK3) and two type II (ActRIIA and BMPR2) BMP receptors. Hemojuvelin (HJV), HFE, and transferrin receptor-2 (TfR2) facilitate this process presumably by forming a plasma membrane complex with BMP receptors. Matriptase-2 (MT2) is a protease and key suppressor of hepatic hepcidin expression and cleaves HJV. Previous studies have therefore suggested that MT2 exerts its inhibitory effect by inactivating HJV. Here, we report that MT2 suppresses hepcidin expression independently of HJV. In Hjv(-/-) mice, increased expression of exogenous MT2 in the liver significantly reduced hepcidin expression similarly as observed in wild-type mice. Exogenous MT2 could fully correct abnormally high hepcidin expression and iron deficiency in MT2(-/-) mice. In contrast to MT2, increased Hjv expression caused no significant changes in wild-type mice, suggesting that Hjv is not a limiting factor for hepcidin expression. Further studies revealed that MT2 cleaves ALK2, ALK3, ActRIIA, Bmpr2, Hfe, and, to a lesser extent, Hjv and Tfr2. MT2-mediated Tfr2 cleavage was also observed in HepG2 cells endogenously expressing MT2 and TfR2. Moreover, iron-loaded transferrin blocked MT2-mediated Tfr2 cleavage, providing further insights into the mechanism of Tfr2's regulation by transferrin. Together, these observations indicate that MT2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway.