Organic anion transporting polypeptides (human: OATPs; rodent: Oatps) were thought to have important functions in bile acid (BA) transport. Oatp1a1, 1a4, and 1b2 are the three major Oatp1 family members in rodent liver. Our previous studies have characterized the BA homeostasis in Oatp1a1-null and Oatp1b2-null mice. The present study investigated the physiological role of Oatp1a4 in BA homeostasis by using Oatp1a4-null mice. Oatp1a4 expression is female-predominant in livers of mice, and thereby it was expected that female Oatp1a4-null mice will have more prominent changes than males. Interestingly, the present study demonstrated that female Oatp1a4-null mice had no significant alterations in BA concentrations in serum or liver, though they had increased mRNA of hepatic BA efflux transporters (Mrp4 and Ostα/β) and ileal BA transporters (Asbt and Ostα/β). In contrast, male Oatp1a4-null mice showed significantly altered BA homeostasis, including increased concentrations of deoxycholic acid (DCA) in serum, liver and intestinal contents. After feeding a DCA-supplemented diet, male but not female Oatp1a4-null mice had higher concentrations of DCA in serum and livers than their WT controls. This suggested that Oatp1a4 is important for intestinal absorption of secondary BAs in male mice. Furthermore, loss of Oatp1a4 function did not decrease BA accumulation in serum or livers of bile-duct-ligated mice, suggesting that Oatp1a4 is not likely a BA uptake transporter. In summary, the present study for the first time demonstrates that Oatp1a4 does not appear to mediate the hepatic uptake of BAs, but plays an important male-predominant role in secondary BA metabolism in mice.