The anaerobic oxidation of methane (AOM) by methanotrophic archaea and sulfate-reducing bacteria is the major sink of methane formed in marine sediments. The study of AOM as well as of methanogenesis in different habitats is essentially connected with the in situ analysis of stable isotope (13C/12C, D/H) signatures (δ-values). For their kinetic interpretation, experimental (cultivation-based) isotope fractionation factors (α-values) are richly available in the case of methanogenesis, but are scarce in the case of AOM. Here we used batch enrichment cultures with high AOM activity and without background methanogenesis from detrital remnants to determine 13C/12C and D/H fractionation factors. The enrichment cultures which originated from three marine habitats (Hydrate Ridge, NE Pacific; Amon Mud Volcano, Mediterranean Sea; NW shelf, Black Sea) were dominated by archaeal phylotypes of anaerobic methanotrophs (ANME-2 clade). Isotope fractionation factors calculated from the isotope signatures as a function of the residual proportion of methane were 1.012–1.039 for 13CH4/12CH4 and 1.109–1.315 for CDH3/CH4. The present values from in vitro experiments were significantly higher than values previously estimated from isotope signature distributions in marine sediment porewater, in agreement with the overlap of other processes with AOM in the natural habitat.