A critical component of the biogeochemical cycle of mercury (Hg) is the transformation of inorganic Hg into neurotoxic monomethylmercury (CH3Hg). Humans are exposed to CH3Hg by consuming marine fish, yet the origin of CH3Hg in fish is a topic of debate. The carbon stable isotopic composition (δ13C) embedded in the methyl group of CH3Hg remains unexplored. This new isotopic information at the molecular level is thought to represent a new proxy to trace the carbon source at the origin of CH3Hg. Here, we present a compound specific stable isotope analysis (CSIA) technique for the determination of the δ13C value of CH3Hg in biological samples by gas chromatography combustion isotope ratio mass spectrometry analysis (GC-C-IRMS). The method consisted first of calibrating a CH3Hg standard solution for δ13C CSIA. This was achieved by comparing three independent approaches consisting of the derivatization and halogenation of the CH3Hg standard solution. The determination of δ13CCH3Hg values on natural biological samples was performed by combining a CH3Hg selective extraction, purification and halogenation followed by GC-C-IRMS analysis. Reference δ13C values were established for a tuna fish certified material (ERM-CE464) originating from the Adriatic Sea (δ13CCH3Hg = -22.1±1.5 ‰, ± 2SD). This value is similar to the δ13C value of marine algal derived particulate organic carbon (δ13CPOC = -21‰).