This study demonstrates that under abiotic dark conditions in aquatic system, humic substances are not only capable of converting Hg(II) to Hg(0) but also able to bind Hg(II) ion. The degree of Hg(II) reduction is significantly influenced by the ratio of -COOH/-OH groups and the sulfur content in the HS, revealing a strong competition between complexation and reduction of Hg(II). This study suggests that abiotic and dark Hg(II) reduction depends on the pH and salinity of aqueous medium. At lower pH (∼ 4.0) and lower salinity (≤ 5.0 PSU), the reduction of Hg(II) to elemental mercury (Hg(0)) was comparatively rapid. Higher -COOH/-OH ratios in HS, favors dark abiotic reduction of Hg(II) as did a lower sulfur (S) content of HS. This study provided a rigorously controlled experimental design that showed that dark abiotic Hg(II) reduction by HS can potentially be important in the aquatic environment and is independent of the photochemical reduction observed in both fresh water and sea water.