Colorectal cancer is one of the most common causes of cancer-related deaths throughout the world. Recently, we reported that proteasome subunit PSMA7 located on 20q13 amplicon was overexpressed and associated with liver metastasis of colorectal cancer. The results indicate that PSMA7 may play an important role in the colorectal cancer progression and provide a unique target site for the development of therapeutic drugs. However, it is unknown how aberrant PSMA7 activation critically regulates the metastatic behavior of colorectal cancer cells. To investigate the role of PSMA7 in the progression of colorectal cancer, we employed the RNA interference technology to knock down the PSMA7 gene in human colon cancer cell line RKO and analyzed its effect and explored the involved mechanisms. Depletion of PSMA7 by shRNA in RKO cells inhibited their anchorage-independent growth and cell invasion and migration. Moreover, PSMA7 depletion was able to strongly suppress the in vivo tumorigenic ability of RKO cells. These effects may be induced by inhibiting CD44 expression directly or indirectly. Genetic or pharmacological inhibition of PSMA7 may therefore be a beneficial strategy in the treatment of colorectal cancer patients.