Monitoring mercury (Hg) levels in biota is considered an important objective for the effectiveness evaluation of the Minamata Convention. While many studies have characterized Hg levels in organisms at multiple spatiotemporal scales, concentration analyses alone often cannot provide sufficient information on the Hg exposure sources and internal processes occurring within biota. Here, we review the decadal scientific progress of using Hg isotopes to understand internal processes that modify the speciation, transport, and fate of Hg within biota. Mercury stable isotopes have emerged as a powerful tool for assessing Hg sources and biogeochemical processes in natural environments. A better understanding of the tissue location and internal mechanisms leading to Hg isotope change is key to assessing its use for biomonitoring. We synthesize the current understanding and uncertainties of internal processes leading to Hg isotope fractionation in a variety of biota, in a sequence of better to less studied organisms (i.e., birds, marine mammals, humans, fish, plankton, and invertebrates). This review discusses the opportunities and challenges of using certain forms of biota for Hg source monitoring and the need to further elucidate the physiological mechanisms that control the accumulation, distribution, and toxicity of Hg in biota by coupling new techniques with Hg stable isotopes.