Antagonistic interactions between mercury (Hg) and selenium (Se), were evaluated in mouse (Mus musculus), as a mammalian model, in a series of controlled exposure experiments. The beneficial effect of Se against Hg toxicity involves a variety of biochemical and toxicological processes that have not been clarified yet. For this purpose, a metallomic workflow based on the use of size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP-MS) detection was complemented with the speciation of selenoproteins and low molecular mass selenium species in serum and liver cytosolic extracts using a multidimensional approach based on SEC-AF-HPLC-ICPMS, using species-unspecific isotope dilution (SUID)-ICP-MS for selenium quantification. The results showed potential interactions between Hg/Se in organs and serum related to accumulation and detoxification processes, in addition to the effects of mercury on selenoproteins in hepatic cytosolic extracts and bloodstream when both elements are administrated at the same time. These results provide information about elements distribution, interactions and homeostasis and reveal the potential of metallomic approaches in exposure experiments.