Reduced graphene oxide (rGO) membranes have huge potential for use in the separation of isotopic water mixtures due to the narrow interlayer spacing forming between the rGO sheets. Although there have been several studies on water purification using graphene oxide (GO) and rGO membranes, no experimental work has been performed towards understanding the role of rGO's oxygen-containing groups in the successful filtration of isotopes. The present work investigates key factors and functional groups that govern the separation performance of rGO membranes. The latter are produced by reducing GO membranes in hydroiodic acid vapor at low temperature (40 °C). The membranes are subsequently tested in filtration experiments using D2O/H2O mixtures and their selectivity and transmembrane flux are recorded as functions of time and temperature. Chemical characterization of the membranes reveals that the hydroxyl and epoxide groups are primarily responsible for the observed selective separation of isotopic water mixtures. Partially reduced GO membranes were found to provide a D2O rejection efficiency that is comparable to their GO counterparts but exhibit higher mechanical robustness that permits longer filtration cycles. Experimental evidence provided herein proves that rGO membranes can be an effective and promising means of isotopic water mixture separation.