Methylmercury (MeHg), a potent neurotoxin, can be formed, migrated and transformed in environmental compartments, accompanying with unique mass-dependent and mass-independent fractionation of mercury (Hg). These Hg isotope fractionation signals have great potential to probe the transformation and transport of MeHg in aquatic environments. However, the majority of studies to date have focused on total Hg isotopic composition, with less attention to the isotopic fractionation of MeHg due to technical difficulties in analysis, which severely hinders the understanding of MeHg isotopic fractionation and its applications. This review a) evaluates the reported analytical methods for Hg isotopic composition of MeHg, including online and offline measurement techniques; b) summarizes the extent and characteristics of Hg isotopic fractionation during MeHg transport and transformation, focusing on methylation, demethylation, trophic transfer and internal metabolism; and c) briefly discusses several applications of MeHg isotopic fractionation signatures in estimating the extent of photodemethylation, tracing the source of Hg species, and diagnosing reaction mechanisms. Additionally, the existing problems and future directions in MeHg isotope fractionation are highlighted to improve the analytical protocol for Hg isotope fractionation and deepen our understanding of Hg isotope fractionation in the biogeochemical cycling of MeHg.