Cardiac microvascular endothelial cells (CMECs) dysfunction induced by hypoxia is an important pathophysiological event in myocardium ischemic injury, whereas, the underlying mechanism is not fully clarified. FoxO transcription factors regulate target genes involved in apoptosis and cellular reactive oxygen species (ROS) production. Therefore, the present study was designed to elucidate the potential role of FoxOs on the hypoxia-induced ROS formation and apoptosis in CMECs. Exposure to low oxygen tension stimulated ROS accumulation and increased apoptosis in CMECs within 6–24 h. Hypoxia also significantly increased the expressions of HIF-1α and FoxO3a. However, hypoxia decreased the phosphorylation of Akt and FoxO3a, correlated with increased nuclear accumulation. Conversely, the expression of FoxO1 was not significantly altered by hypoxia. After inhibition of HIF-1α by siRNA, we observed that hypoxia-induced ROS accumulation and apoptosis of CMECs were decreased. Meanwhile, knockdown of HIF-1α also inhibited hypoxia induced FoxO3a expression in CMECs, but did not affect FoxO1 expression. Furthermore, hypoxia-induced ROS formation and apoptosis in CMECs were correlated with the disturbance of Bcl-2 family proteins, which were abolished by FoxO3a silencing with siRNA. In conclusion, our data provide evidence that FoxO3a leads to ROS accumulation in CMECs, and in parallel, induces the disturbance of Bcl-2 family proteins which results in apoptosis.