BACKGROUND AND PURPOSE The human organic anion transporting polypeptide 1A2 (OATP1A2) is expressed in cells from several regions of the human body, including the kidney, cholangiocytes and the blood‐brain barrier, and mediates the cellular flux of various anionic substances, including drugs in clinical use. Several related mammalian transporters have been shown to be subject to post‐translational regulation, including kinase‐induced internalization. In the present study the role of protein kinase C (PKC) in the regulation of OATP1A2 was investigated in an in vitro cell model. EXPERIMENTAL APPROACH COS‐7 cells in which OATP1A2 was overexpressed were treated with the PKC‐specific activator (phorbol 12‐myristate 13‐acetate; PMA) and the PKC‐specific inhibitor (Go6976). The impact of these treatments on the function and regulation of OATP1A2 was determined. KEY RESULTS PKC activation decreased the transport function of OATP1A2 in a time‐ and concentration‐dependent manner. PMA (0.1 µM) decreased the Vmax of oestrone‐3‐sulphate uptake and decreased the cell surface expression of OATP1A2 immunoreactive protein; these effects of PMA were prevented by the PKC specific inhibitor Go6976. In further studies, PMA treatment accelerated the internalization of OATP1A2 but did not affect its recycling. The disruption of clathrine‐dependent endocytosis attenuated both the constitutive and PKC‐modulated internalization of OATP1A2. In contrast, blocking the caveolin‐dependent pathway was without effect. CONCLUSIONS AND IMPLICATIONS PKC regulates the transport function of OATP1A2 by modulating protein internalization; this effect of PKC is mediated in part by clathrine‐dependent pathways.