OATP1B3 is a member of the OATP (organic anion transporting polypeptides) superfamily, responsible for mediating the transport of numerous endogenous and xenobiotic substances. Although initially reported to be exclusively expressed in the liver, several studies reported that OATP1B3 is frequently expressed in multiple types of cancers and may be associated with differing clinical outcomes. However, a detailed investigation on the expression and function of OATP1B3 protein in cancer has been lacking. In this study, we confirmed that colon and pancreatic cancer cells express variant forms of OATP1B3, different from OATP1B3 wild-type (WT) expressed in the normal liver. OATP1B3 variant 1 (V1), the most prevalent form among the variants, contains alternative exonic sequences (exon 2a) instead of exons 1 and 2 present in OATP1B3 WT. The translated product of OATP1B3 V1 is almost identical to OATP1B3 WT, with exception to the first 28 amino acids at the N-terminus. Exogenous expression of OATP1B3 V1 revealed that OATP1B3 V1 undergoes post-translational modifications and proteasomal degradation to a differing extent compared to OATP1B3 WT. OATP1B3 V1 showed only modest transport activity toward cholecystokin-8 (CCK-8, a prototype OATP1B3 substrate) in contrast to OATP1B3 WT showing a markedly efficient uptake of CCK-8. Consistent with these results, OATP1B3 V1 was localized mainly in the cytoplasm with a much lower extent of trafficking to the surface membrane compared to OATP1B3 WT. In summary, our results demonstrate that colon and pancreatic cancer cells express variant forms of OATP1B3 with only limited transport activity and different subcellular localization compared to OATP1B3 WT. These observed differences at the molecular and functional levels will be important considerations for further investigations of the biological and clinical significance of OATP1B3 expression in cancer.