Solute carrier organic anion transporter family member 1B3 (SLCO1B3) is a gene that encodes an organic anion-transporting polypeptide (OATP) 1B3, a membrane-bound multi-specific transporter in hepatocytes. SLCO1B3 was first reported in hepatocytes. Later, it was found that its expression is higher in colorectal cancer (CRC) than in the adjacent normal tissue. However, the role of SLCO1B3 in CRC is not well elucidated. In this study, the correlation between SLCO1B3 and the overall survival (OS) of CRC patients was evaluated using data from the GEO database. This study evaluated the relationship between SLCO1B3 and the clinicopathological characteristics and prognosis of CRC patients. The effects of SLCO1B3 knockdown, on human CRC cell proliferation, migration, and invasion in vitro and CRC tumorigenesis and metastasis in vivo were also examined. In addition, next-generation sequencing was used to identify SLCO1B3 mediators. The results confirmed the association between SLCO1B3 and poor OS of CRC patients, and SLCO1B3 was identified as the top hub gene associated with the OS. The study showed that high SLCO1B3 expression was associated with poor tumor differentiation, advanced disease stage, tumor invasion, lymph node metastasis, and poor OS. Next-generation sequencing revealed that SLCO1B3 knockdown affected the expression of several genes involved in cancer invasion, metastasis, and DNA repair. Moreover, the western blot analysis showed that SLCO1B3 knockdown downregulated p-STAT3, MMP-2, and MMP-9. In summary, we demonstrated that SLCO1B3 acts as a novel carcinogen in the CRC that drives the CRC tumorigenesis and metastasis. SLCO1B3 inhibitors, alone or in combination with current drugs, may have therapeutic benefits in CRC.