Dehydrochlorination is one of the main (thus far discovered) processes for aerobic microbial transformation of hexachlorocyclohexane (HCH) which is mainly catalyzed by LinA enzymes. In order to gain a better understanding of the reaction mechanisms, multi-element compound-specific stable isotope analysis was applied for evaluating αand γ-HCH transformations catalyzed by LinA1 and LinA2 enzymes. The isotopic fractionation (ε E ) values for particular elements of (), whereas the dual-isotope fractionation patterns were almost identical for both enantiomers (Λ C−Cl = 2.4 ± 0.4 and 2.5 ± 0.2, Λ H−C = 12.9 ± 2.4 and 14.9 ± 1.1). The ε E of γ-HCH transformation by LinA1 and LinA2 were −7.8 ± 1.0‰ and −7.5 ± 0.8‰ (ε C ), −2.7 ± 0.3‰ and −2.5 ± 0.4‰ (ε Cl ), −170 ± 25‰ and −150 ± 13‰ (ε H ), respectively. Similar Λ C−Cl values (2.7 ± 0.2 and 2.9 ± 0.2) were observed as well as similar Λ H−C values (20.1 ± 2.0 and 18.4 ± 1.9), indicating a similar reaction mechanism by both enzymes during γ-HCH transformation. This is the first data set on 3D isotope fractionation of αand γ-HCH enzymatic dehydrochlorination, which gave a more precise characterization of the bond cleavages, highlighting the potential of multi-element compound-specific stable isotope analysis to characterize different transformation processes (e.g., dehydrochlorination and reductive dehalogenation).