To date, the aerobic biodegradation of hexabromocyclododecanes (HBCDs) by HBCD-degrading bacteria has largely been studied via a mixture of all diastereoisomers. To improve the understanding of the degradation process of different diastereoisomers, we established an optimized method for isolating three diastereoisomers (alpha-, beta-, and gamma-HBCD) by high-performance liquid chromatography (HPLC) with a C18 column. One pure bacterial strain, Pseudomonas sp. strain GJY, was isolated with excellent HBCD biodegradation activity. At 30 degrees C, pH 7.0, and an initial HBCD concentration of 1 mg/L, the biodegradation rates of alpha-, beta-, and gamma-HBCDs were 85.38%, 82.64%, and 75.50% after 8 days of aerobic incubation, respectively. Only the enrichment of (+) gamma-HBCD was observed, and no enantioselectivity was found in alpha- and beta-HBCD. Finally, the metabolites were determined from a full scan, precursor ion scan, and product ion scan by UPLC-MS/MS. PBCDe, TeBCDe, DBCDi, PBCD-ols, TeBCDe-ols, and TrBCD-triols were the main products, and they exhibited differences in the three diastereoisomers. Interestingly, no aerobic hydroxylation product of HBCD, such as HBCD-ols and HBCD-diols, was identified in any of the degradation processes. In consideration of these products, the main degradation pathways of the three diastereoisomers were proposed.