Human concentrative nucleoside transporter1 (hCNT1), a member of the solute carrier 28 family, is a high-affinity transporter of anti-cancer nucleoside drugs (e.g. gemcitabine). While hCNT1 is implicated in sensitizing solid tumors to gemcitabine, its role in determining gemcitabine efficacy in human ovarian cancers remains unknown. Here we examined the functional expression of hCNT1 in normal and cancerous ovarian cells and compared its contributions towards gemcitabine efficacy in histological subtypes of ovarian cancer. RNA and protein analysis identified that unlike normal ovarian surface epithelial cells (IOSE80), which expressed high levels of hCNT1 at the cell surface, hCNT1 expressions were diminished in ovarian cancer cell lines. Consistently, 3H-gemcitabine transports in ovarian cancer cells were reduced by 3-10 fold compared with that of IOSE80 cells. Stable retroviral expression of hCNT1 in various ovarian cancer cell lines displayed variations in hCNT1 localizations, 3H-gemcitabine transports, and drug sensitivities. hCNT1-expressing endometroid cancer cells exhibited the highest sensitivity to gemcitabine treatment (~140 fold IC50 decrease). hCNT1-expressing serous ovarian cancer cells (OV90) displayed a moderate level of sensitivity (~25 fold IC50 decrease) that was ~2000-fold higher than that observed with clear cell carcinoma (CCC; ES-2 and TOV21G) and teratocarcinoma cells (PA-1). hCNT1 was not recruited to the cell surface in teratocarcinoma cells and was mistargeted to golgi in CCC cells. These data show that hCNT1 transportability is highly diminished in human ovarian cancers and the reintroduction of hCNT1 can selectively regain gemcitabine sensitivity in the endometroid and serous subtypes but not in the teratocarcinoma or CCC subtypes.