Cancer-type Organic anion transporting polypeptide 1B3, ct-OATP1B3-V1 is a tumor-specific isoform of liver-type OATP1B3 (Lt-OATP1B3). Ct-OATP1B3-V1 is identical with liver-specific Lt-OATP1B3 except it lacks the first 28 amino acids. Although there is a growing interest in using this isoform as a biomarker for colorectal cancer, available data regarding cellular localization and function of ct-OATP1B3-V1 remains controversial. The main objective of our study was to clarify the localization and function of ct-OATP1B3-V1 in vitro and in vivo, and to investigate its role in chemotherapy sensitivity. For this aim, A431 and HCT-8 carcinoma cell lines overexpressing ct-OATP1B3-V1 were generated. With the help of these cell lines, localization and activity of ct-OATP1B3-V1 as well as its effect on chemotherapy sensitivity was examined both in vitro and in vivo. We found that ct-OATP1B3-V1 is a functional plasma membrane transporter that sensitizes the cells toward various chemotherapeutics, including docetaxel, oxaliplatin and capecitabine metabolites in vitro. Increased sensitivity to docetaxel and capecitabine of ct-OATP1B3-V1 expressing cells was also confirmed in in vivo experiments performed on A431-V1 derived xenografts. However, due to the apparent proliferative advantage of V1-expressing xenografts over the mock-transfected control, they could not be completely eradicated by either docetaxel or capecitabine treatment. Our results demonstrate that while ct-OATP1B3-V1 can be exploited to inhibit tumor growth, this strategy alone is likely insufficient for complete tumor elimination, possibly due to the more complex in vivo functions of ct-OATP1B3-V1.