Glyoxalase 2 (Glo2) is the second enzyme in the glyoxalase system, and while its cytoplasmic function is well-established, its subcellular localization in the nucleus and potential role there remain unexplored. This study investigates nuclear localization and function of Glo2 in human breast cancer (MCF7) and normal fibroblast-derived (HDF) cells. Glo2 was present in both cytoplasm and nucleus of MCF7 and HDF cells. Interestingly, Glo2 expression levels were significantly higher in MCF7 compared to HDF, especially when considering the nuclear Glo2. Western-blot analysis revealed the monomeric 29 KDa Glo2 form and a 47 KDa Glo2 form suggesting complex formation with other proteins in the cytoplasm of MCF7 and HDF cells. Interestingly, at the nuclear level only the tumor cells showed the 47 KDa Glo2 form. Sequential salt extraction indicated a potential association of Glo2 with non-histone proteins. Intriguingly, nuclear Glo2 positively correlated with cell cycle proliferative phase (G2/M), more markedly in MCF7. Furthermore, higher levels of S-glutathionylation of nuclear proteins were observed in MCF7 compared to HDFs, and “in vitro” addition of Glo2 to nuclear extracts increased S-glutathionylation levels, significantly in MCF7. Finally, inhibiting Glo2 increased doxorubicin cytotoxicity in MCF7. Overall, these results indicate that nuclear Glo2 may play a role in in promoting cancer cell survival and targeting Glo2 could be a promising strategy to improve the efficacy of some anticancer therapies in breast cancer.