Kinetic isotopic fractionation of carbon and nitrogen during RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) biodegradation was investigated with pure bacterial cultures under aerobic and anaerobic conditions. Relatively large bulk enrichments in ¹⁵N were observed during biodegradation of RDX via anaerobic ring cleavage (ε¹⁵N = −12.7‰ ± 0.8‰) and anaerobic nitro reduction (ε¹⁵N = −9.9‰ ± 0.7‰), in comparison to smaller effects during biodegradation via aerobic denitration (ε¹⁵N = −2.4‰ ± 0.2‰). ¹³C enrichment was negligible during aerobic RDX biodegradation (ε¹³C = −0.8‰ ± 0.5‰) but larger during anaerobic degradation (ε¹³C = −4.0‰ ± 0.8‰), with modest variability among genera. Dual-isotope ε¹³C/ε¹⁵N analyses indicated that the three biodegradation pathways could be distinguished isotopically from each other and from abiotic degradation mechanisms. Compared to the initial RDX bulk δ¹⁵N value of +9‰, δ¹⁵N values of the NO₂⁻ released from RDX ranged from −7‰ to +2‰ during aerobic biodegradation and from −42‰ to −24‰ during anaerobic biodegradation. Numerical reaction models indicated that N isotope effects of NO₂⁻ production were much larger than, but systematically related to, the bulk RDX N isotope effects with different bacteria. Apparent intrinsic ε¹⁵N-NO₂⁻ values were consistent with an initial denitration pathway in the aerobic experiments and more complex processes of NO₂⁻ formation associated with anaerobic ring cleavage. These results indicate the potential for isotopic analysis of residual RDX for the differentiation of degradation pathways and indicate that further efforts to examine the isotopic composition of potential RDX degradation products (e.g., NO_x) in the environment are warranted.