Hexabromocyclododecane (HBCD), a flame retardant classified as a Persistent Organic Pollutant (POP), undergoes stereoisomer-specific microbial transformation with significant environmental and health implications. However, the underlying mechanisms of this stereoisomer-specific microbial transformation remain poorly understood. In this study, high-purity HBCD chiral isomers were isolated using an optimized high-performance liquid chromatography (HPLC) method and their transformation by Acinetobacter hemolyticum sp. strain HW-2 was investigated through transcriptomic analysis. Within three days, strain HW-2 removed (+) α-, (-) α-, (+) β-, (-) β-, (+) γ-, and (-) γ-HBCD with respective removal efficiencies of 52.38 %, 71.08 %, 71.07 %, 63.34 %, 47.47 %, and 77.05 %. Transcriptomic data revealed stereoisomer-specific processes in HBCD transport, response, and transformation. Strain HW-2 upregulated major facilitator superfamily (MFS) transport genes for HBCD uptake, with distinct genes activated for different diastereoisomers. Compared to γ-HBCD, α- and β-HBCD exerted greater stress on strain HW-2, leading to increased expression of efflux genes and antioxidant-related genes. The transformation of HBCD stereoisomers involved distinct functional enzymes, with only (-) γ-HBCD metabolized via the aromatic compound metabolic pathway. This study elucidates the stereoisomeric-specific transformation mechanisms underlying HBCD transformation by strain HW-2, offering valuable insights for theoretical and practical applications in HBCD remediation.