Rhomboid domain containing 1 (RHBDD1), is a member of the rhomboid protease family, which has a pivotal role in the progression of numerous severe malignancies. However, its role in colorectal carcinoma (CRC) remains to be elucidated. In the present study, RHBDD1 was shown to be widely expressed in CRC cell lines. Lentivirus-mediated RNA interference was employed to knockdown RHBDD1 expression in RKO CRC cells. Functional analyses indicated that depletion of RHBDD1 expression resulted in significantly reduced CRC cell proliferation and colony formation, and induced a G0/G1 phase cell cycle arrest. The findings of the present study suggest that RHBDD1 may contribute to CRC tumorigenesis and serve as a potential therapeutic target in human CRC.