Almadén, Spain, is the world's largest mercury (Hg) mining district, which has produced over 250,000 metric tons of Hg representing about 30% of the historical Hg produced worldwide. The objective of this study was to measure Hg isotopic compositions of cinnabar ore, mine waste calcine (retorted ore), elemental Hg (Hg⁰(L)), and elemental Hg gas (Hg⁰(g)), to evaluate potential Hg isotopic fractionation. Almadén cinnabar ore δ²⁰²Hg varied from − 0.92 to 0.15‰ (mean of − 0.56‰, σ = 0.35‰, n = 7), whereas calcine was isotopically heavier and δ²⁰²Hg ranged from − 0.03‰ to 1.01‰ (mean of 0.43‰, σ = 0.44‰, n = 8). The average δ²⁰²Hg enrichment of 0.99‰ between cinnabar ore and calcines generated during ore retorting indicated Hg isotopic mass dependent fractionation (MDF). Mass independent fractionation (MIF) was not observed in any of the samples in this study. Laboratory retorting experiments of cinnabar also were carried out to evaluate Hg isotopic fractionation of products generated during retorting such as calcine, Hg⁰(L), and Hg⁰(g). Calcine and Hg⁰(L) generated during these retorting experiments showed an enrichment in δ²⁰²Hg of as much as 1.90‰ and 0.67‰, respectively, compared to the original cinnabar ore. The δ²⁰²Hg for Hg⁰(g) generated during the retorting experiments was as much as 1.16‰ isotopically lighter compared to cinnabar, thus, when cinnabar ore was roasted, the resultant calcines formed were isotopically heavier, whereas the Hg⁰(g) generated was isotopically lighter in Hg isotopes.