Dichlorprop [(R,S)-2-(2,4-dichlorophenoxy)propanoic acid], a chiral herbicide belonging to phenoxyalkanoic acid herbicides, is extensively used around the world, and is frequently detected in various habitats. Although (S)-dichlorprop has no herbicidal activity, its toxicity and ecological risk are higher than (R)-dichlorprop. In our previous studies, the isolated strain Sphingopyxis sp. DBS4 was confirmed to mineralize both (R)-dichlorprop and (S)-dichlorprop, but the key genes responsible for the initial degradation of (R)-/(S)-dichlorprop were still unclear. Here, based on genome sequencing and enzyme purification, a new dioxygenase gene, spoA, responsible for the initial transformation of (S)-dichlorprop to 2,4-dichlorophenol and pyruvate was cloned from the rac-dichlorprop-utilizing strain of Sphingopyxis sp. DBS4. SpoA, an α-ketoglutarate-dependent dioxygenase, exhibited low amino acid sequence identity to the reported (S)-dichlorprop-transforming dioxygenase SdpA (34.8%), and showed enantioselective activity toward (S)-enantiomers of dichlorprop and mecoprop, but no activity toward the corresponding (R)-enantiomers. The K_m value of SpoA for (S)-dichlorprop was 134.3 μM, and the k_cat/K_m was determined to be 7.5 × 10⁻² μM⁻¹ s⁻¹. Molecular docking and site-directed mutagenesis showed that two residues of SdpA, Lys101 and Asp119, are the key sites for the enantioselective transformation of (S)-dichlorprop. Our study identifies a new dioxygenase for the enantioselective transformation of (S)-dichlorprop and deepens our understanding of the microbial catabolism of chiral phenoxyalkanoic acid herbicides.