Arsenic is a toxic metalloid that is widely distributed in the environment, and its toxicity has been demonstrated in several models. However, the mechanism of arsenic toxicity still remains unclear. In this study, the toxic effects of sodium arsenite (1–7 mM) on yeast cells were investigated. The experimental results showed that sodium arsenite inhibited yeast cell growth, and the inhibitory effect of cell growth (OD600 nm values) was positively correlated with arsenite concentrations. Sodium arsenite caused loss of cell viability in a concentration- and duration-dependent manner in yeast cells. However, arsenite-caused cell viability loss was blocked by either antioxidants (200 U mL−1 CAT and 0.5 mM AsA) or Ca2+ antagonists (0.5 mM LaCl3 and 0.5 mM EGTA). We also found intracellular reactive oxygen species (ROS) and Ca2+ levels increased significantly in yeast cells after exposure to 3 mM and 7 mM sodium arsenite for 6 h compared with the control. These results indicated that high concentrations of arsenite-induced yeast cell killing was associated with elevated levels of intracellular ROS and Ca2+.