Green S (GRS) is widely used as a food colorant and a pharmaceutical excipient, however, its genotoxic potential is questionable and to our knowledge no comprehensive study on its genotoxic effects has been conducted. The aim of this study was to investigate the genotoxic effects of GRS in vitro in combination with molecular docking simulations with DNA and protein targets. For this purpose, human peripheral lymphocytes (HPLs), pBR322 DNA, Salmonella TA98 and TA100 strains were exposed to GRS (25, 50, and 100 mu g/mL) for 24 and 48 hr. Consequently, the micronucleus (MN) induction, DNA damage/cleavage/protection potential and reverse mutation in Salmonella typhimurium were studied. Moreover, affinity of GRS to bind with DNA and alpha-beta tubulin was demonstrated using AutoDock 4.2. GRS significantly increased the MN frequency at 24 (p < 0.05) and 48 (p < 0.001) hr treatment in HPLs. Notably, the increase in MN frequency at 48-hr was time-dependent (p < 0.05 - p < 0.001). However, GRS was unable to induce DNA damage in the comet assay and neither demonstrated a cleavage of pBR322 DNA nor protected against H202. Also, GRS did not increase the reverse mutation frequency in TA98 and TA100 strains. Molecular docking analysis suggested that while GRS may weakly interact with DNA (Delta G(best) = -3.59), it showed strong binding affinity to alpha- (Delta G(best) = 6.95) and beta-tubulin (Delta G(best) = -5.03) subunits. When the MN and molecular docking results are evaluated together, our results suggest carcinogenicity in human HPLs due to noncovalent interactions with tubulin proteins. Therefore, the end user should be more aware of the possible health risks that may arise from exposure to this food colorant and its use should to be restricted.