Aldo-keto reductase 1B1 and 1B3 (AKR1B1 and AKR1B3) are the primary human and mouse prostaglandin F2α (PGF2α) synthases, respectively, which catalyze the NADPH-dependent reduction of PGH2, a common intermediate of various prostanoids, to form PGF2α. In this study, we found that AKR1B1 and AKR1B3, but not AKR1B7 and AKR1C3, also catalyzed the isomerization of PGH2 to PGD2 in the absence of NADPH or NADP+. Both PGD2 and PGF2α synthase activities of AKR1B1 and AKR1B3 completely disappeared in the presence of NADP+ or after heat treatment of these enzymes at 100 °C for 5 min. The Km, Vmax, pK and optimum pH values of the PGD2 synthase activities of AKR1B1 and AKR1B3 were 23 and 18 μm, 151 and 57 nmol·min−1·(mg protein)−1, 7.9 and 7.6, and pH 8.5 for both AKRs, respectively, and those of PGF2α synthase activity were 29 and 33 μm, 169 and 240 nmol·min−1·(mg protein)−1, 6.2 and 5.4, and pH 5.5 and pH 5.0, respectively, in the presence of 0.5 mm NADPH. Site-directed mutagenesis of the catalytic tetrad of AKR1B1, composed of Tyr, Lys, His and Asp, revealed that the triad of Asp43, Lys77 and His110, but not Tyr48, acts as a proton donor in most AKR activities, and is crucial for PGD2 and PGF2α synthase activities. These results, together with molecular docking simulation of PGH2 to the crystallographic structure of AKR1B1, indicate that His110 acts as a base in concert with Asp43 and Lys77 and as an acid to generate PGD2 and PGF2α in the absence of NADPH or NADP+ and in the presence of NADPH, respectively.