<jats:title>Abstract</jats:title><jats:p>Prenylated indole alkaloids featuring spirooxindole rings possess a 3<jats:italic>R</jats:italic> or 3<jats:italic>S</jats:italic> carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3<jats:italic>R</jats:italic> or 3<jats:italic>S</jats:italic>-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3<jats:italic>S</jats:italic>-spirooxindole construction in the biosynthesis of 21<jats:italic>R</jats:italic>-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3<jats:italic>S</jats:italic>-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3<jats:italic>R</jats:italic>-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3<jats:italic>S</jats:italic> spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.