Compound-specific isotope analysis (CSIA) can provide insights into the natural attenuation processes of hexachlorocyclohexanes (HCHs), an important class of persistent organic pollutants. However, the interpretation of HCH stable isotope fractionation is conceptually challenging. HCHs exist as different conformers that can be converted into each other, and the enzymes responsible for their transformation discriminate among those HCH conformers. Here, we investigated the enzyme specificity of apparent 13C- and 2H-kinetic isotope effects (AKIEs) associated with the dehydrochlorination of γ-HCH (lindane) by two variants of the lindane dehydrochlorinases LinA1 and LinA2. While LinA1 and LinA2 attack γ-HCH at different trans-1,2-diaxial H–C–C–Cl moieties, the observed C and H isotope fractionation was large, typical for bimolecular eliminations, and was not affected by conformational mobility. 13C-AKIEs for transformation by LinA1 and LinA2 were the same (1.024 ± 0.001 and 1.025 ± 0.001, respectively), whereas 2H-AKIEs showed minor differences (2.4 ± 0.1 and 2.6 ± 0.1). Variations of isotope effects between LinA1 and LinA2 are small and in the range reported for different degrees of C–H bond cleavage in transition states of dehydrochlorination reactions. The large C and H isotope fractionation reported here for experiments with pure enzymes contrasts with previous observations from whole cell experiments and suggests that specific uptake processes by HCH-degrading microorganisms might modulate the observable HCH isotope fractionation at contaminated sites.