In this work we assessed the potential for the denitrification linked to methanol consumption in a microbial community inhabiting the top layer of the sediment of a pristine lake, Lake Washington in Seattle. Stable isotope probing with 13C methanol was implemented in near in situ conditions and also in the presence of added nitrate. This revealed that the bacterial population involved in methanol uptake was dominated by species belonging to the Methylophilaceae, most prominently species belonging to the genus Methylotenera. Based on relative abundance of specific phylotypes in DNA clone libraries generated from 13C labelled DNA, some of these species appear not to require nitrate to assimilate methanol while others assimilate methanol in a nitrate-dependent fashion. A pure culture of Methylotenera mobilis strain JLW8 previously isolated from the same study site was investigated for denitrification capability. This culture was demonstrated to be able to grow on methanol when nitrate was present, in aerobic conditions, while in media supplemented with ammonium it did not grow on methanol. The denitrifying capability of this strain was further demonstrated in defined laboratory conditions, by measuring accumulation of N2O. This study provides new insights into the potential involvement of Methylophilaceae in global nitrogen cycling in natural environments and highlights the connection between global carbon and nitrogen cycles.