The objective of this work was to determine the magnitude of mercury isotope fractionation during volatilization of Hg(0) dissolved in aqueous solution to the gas phase and to develop a method for the precise determination of isotope ratios of gaseous Hg(0) by multi-collector ICP-MS. Hg(0) was generated in situ by reducing Hg(II) using SnCl2 and subsequently purged into a trapping solution. A 316 μM KMnO4 trapping solution, acidified by 0.9 M sulfuric acid, recovered 96.1 ± 4.0% of Hg (2 SD, n = 20). The observed δ202Hg of −0.049 ± 0.065‰ (2 SD, n = 17) relative to the initial Hg standard was not greater than the daily external precision of the isotope ratio measurement, which is typically <0.100‰ (2 SD). The volatilization experiments were conducted by reducing Hg(II) completely using SnCl2, letting the developed Hg(0) evaporate from solution and purging the Hg(0) vapor in the headspace over the solution into the trapping solutions. The concentration and isotope ratios of Hg in both the trapping solutions and the Hg(0) remaining in solution were measured at different time intervals. The kinetics of the volatilization was found to be a first order process. The pattern of isotope fractionation during volatilization followed a Rayleigh fractionation with an observed maximum δ202Hg value of 1.48 ± 0.07‰ (2 SD). Fractionation factors of 1.00044 and 1.00047 were obtained in two independent experiments. These results provide the first experimental proof for, and quantified the pattern and magnitude of, the Hg isotope fractionation during volatilization of Hg(0) from solution into the gas phase.