Methylotrophs play an essential role in the global carbon cycle due to their participation in methane oxidation and C1 metabolism. Despite this important biogeochemical role, marine and estuarine microorganisms that consume C1 compounds are poorly characterized. In this study, we investigated the diversity of active methylotrophs and methanotrophs in sediment from the Colne Estuary (Brightlingsea, UK). Aerobic surface sediment samples were examined for the presence of C1-utilizing communities using DNA stable-isotope probing (DNA-SIP) with 13C-labelled methane, methanol and monomethylamine. Active methylotrophic bacteria were confirmed after DNA-SIP and denaturing gradient gel electrophoresis analyses. Clone libraries of 16S rRNA gene amplicons revealed the presence of methylotrophic bacteria affiliated with Methylophaga spp. in methanol and monomethylamine incubations. The addition of marine ammonium mineral salts medium to the microcosms increased the rate of substrate metabolism in DNA-SIP incubations, although nutrient addition did not affect the active populations contributing 13C-labelled DNA. The 13CH4 SIP incubations indicated the predominant activity of type I methanotrophs and microarray hybridization of amplified particulate methane monooxygenase (pmoA) genes confirmed the role of type Ia methanotrophs in SIP incubations. Type II methanotrophs (i.e. Methylocystis and Methylosinus) were only detected in the original sediment and in the unlabelled DNA fractions, which indicated that type II methanotrophs were not actively involved in C1 compound assimilation in DNA-SIP incubations with estuarine surface sediment samples.