Both zinc (Zn²⁺) and reactive oxygen species (ROS) have been shown to accumulate during hypoxic-ischemic stress and play important roles in pathological processes. Here we studied Zn²⁺ and ROS accumulations by employing fluorescent probes in HeLa cells to further the understanding of cause and effect relationship of these two important cellular signaling during chemical-ischemia, stimulated by oxygen and glucose deprivation (OGD). We observed two Zn²⁺ rises that were divided into four phases in the course of 30 minutes OGD. The first Zn²⁺ rise was a transient, which was followed by a latent phase during which Zn²⁺ levels recovered, however remained above a basal level. The final phase was the second Zn²⁺ rise that reached a sustained plateau called Zn²⁺ overload. Zn²⁺ rises were not observed when were treated by TPEN (a Zn²⁺ chelator) or thapsigargin (depleting Zn²⁺ from intracellular stores), indicating that Zn²⁺ originated from intracellular stores. Damaging mitochondria with FCCP significantly reduced the second Zn²⁺ rise, indicating that the mitochondrial Zn²⁺ accumulation contributes to Zn²⁺ overload. We also detected two OGD-induced ROS rises. Two Zn²⁺ rises preceded two ROS rises. Removal of Zn²⁺ reduced or delayed OGD- and FCCP-induced ROS generation, indicating that Zn²⁺ contributes to mitochondrial ROS generation. There was a Zn²⁺-induced increase in functional component of NADPH oxidase, p47phox, thus suggesting that NADPH oxidase may mediate Zn²⁺-induced ROS. We suggest a new mechanism of crosstalk between Zn²⁺ and mitochondrial ROS through positive feedback processes that eventually causes excessive free Zn²⁺ and ROS accumulations during the course of ischemic-stress.