Deposition, remodeling, and signaling of the extracellular matrix facilitate tumor growth and metastasis. Here, we demonstrated that an enzyme, collagen prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), which is involved in collagen synthesis and deposition, had elevated expression in colorectal cancers (CRCs) as compared to normal colonic tissues. The expression of P4HA1 in CRCs was independent of patient's age, race/ethnicity, gender, pathologic stage and grade, tumor location, and microsatellite instability (MSI) and p53 status. By modulating P4HA1 with shRNA, there was a reduction in malignant phenotypes of CRCs, including cell proliferation, colony formation, invasion, migration, and tumor growth, in mice regardless of their p53 and MSI status. Immunoblot analysis of excised xenograft tumors developed from cells with silenced PH4HA1 showed low levels of proliferating cell nuclear antigen. Further, in CRC mouse models, silencing of P4HA1 in HT29 cells resulted in less metastasis to liver and bone. P4HA1 expression was regulated by miR-124, and inhibition of cell growth was noted for CRC cells treated with miR-124. Furthermore, low levels of the transcriptional repressor EZH2 reduced P4HA1 expression in CRC cells. Inhibition of P4HA1 with the small molecule inhibitor diethyl-pythiDC decreased AGO2 and MMP1, which are P4HA1 target molecules, and reduced the malignant phenotypes of CRC cells. Treatment of CRC patient-derived xenografts that exhibit high expression of P4HA1 with diethyl-pythiDC resulted in tumor regression. Thus, the present study shows that P4HA1 contributes to CRC progression and metastasis and that targeting of P4HA1 with diethyl-pythiDC could be an effective therapeutic strategy for aggressive CRCs.