Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. hOAT4 is abundantly expressed in the placenta. In the current study, we examined the regulation of hOAT4 by pregnancy-specific hormones progesterone (P4) and 17β-estradiol (E2) and by protein kinase C (PKC) in human placental BeWo cells. P4 induced a time- and concentration-dependent down regulation of hOAT4 transport activity, whereas E2 had no effect on hOAT4 function. The down regulation of hOAT4 activity by P4 mainly resulted from a decreased cell surface expression without a change in total cell expression of the transporter, kinetically revealed as a decreased Vmax without significant change in Km. Activation of PKC by phorbol 12,13-dibutyrate (PDBu) also resulted in an inhibition of hOAT4 activity through a decreased cell surface expression of the transporter. However, P4-induced down regulation of hOAT4 activity could not be prevented by treating hOAT4-expressing cells with PKC inhibitor, staurosporine. We concluded that both P4 and activation of PKC inhibited hOAT4 activity through redistribution of the transporter from cell surface to the intracellular compartments. However, P4 regulates hOAT4 activity by mechanisms independent of PKC pathway.