Abstract Nickel (Ni) is an essential yet toxic trace element. Although a cofactor for many metalloenzymes, nickel function and metabolism is not fully explored in eukaryotes. Molecular biology and metallomic methods were utilized to explore the new physiological functions of nickel in Saccharomyces cerevisiae. Here we showed that MTM1 knockout cells displayed much stronger nickel tolerance than wild-type cells and mitochondrial accumulations of Ni and Fe of mtm1Δ cells dramatically decreased compared to wild-type cells when exposed to excess nickel. Superoxide dismutase 2 (Sod2p) activity in mtm1Δ cells was severely attenuated and restored through Ni supplementation in media or total protein. SOD2 mRNA level of mtm1Δ cells was significantly higher than that in the wild-type strain but was decreased by Ni supplementation. MTM1 knockout afforded resistance to excess nickel mediated through reactive oxygen species levels. Meanwhile, additional Ni showed no significant effect on the localization of Mtm1p. Our study reveals the MTM1 gene plays an important role in nickel homeostasis and identifies a novel function of nickel in promoting Sod2p activity in yeast cells.