Acquisition and distribution of zinc supports a number of biological processes. Various molecular factors are involved in zinc metabolism but not fully explored.Spontaneous mutants were generated in yeast with excess zinc culture followed by whole genome DNA sequencing to discover zinc metabolism related genes by bioinformatics. An identified mutant was characterized through metallomic and molecular biology methods.Here we reported that MTM1 knockout cells displayed much stronger zinc tolerance than wild type cells on SC medium when exposed to excess zinc. Zn accumulation of mtm1Δ cells was dramatically decreased compared to wild type cells under excessive zinc condition due to MTM1 deletion reduced zinc uptake. ZRC1 mRNA level of mtm1Δ cells was significantly higher than that in the wild-type strain leading to increased vacuolar zinc accumulations in mtm1Δ cells. The mRNA levels of ZRT1 and ZAP1 decreased in mtm1Δ cells contributing to less Zn uptake. The zrc1Δmtm1Δ double knockout strain exhibited Zn sensitivity. MTM1 knockout did not afford resistance to excess zinc through an effect mediated through an influence on levels of ROS. Superoxide dismutase 2 (Sod2p) activity in mtm1Δ cells was severely impaired and not restored through Zn supplementation. Meanwhile, additional Zn showed no significant effect on the localization and expression of Mtm1p.Our study reveals the MTM1 gene plays an important role in the regulation of zinc homeostasis in yeast cells via changing zinc uptake and distribution. This discovery provides new insights for better understanding biochemical communication between vacuole and mitochondrial in relation to zinc-metabolism.