The present study was performed to determine the protective effect of Zinc on the rat testicular ischemia-reperfusion (I/R) injury and its mechanism. In vivo, the pathological changes and the apoptosis index were significantly relieved in the rats with Low-dose Zinc pretreatment, compared to the I/R group. After Low-dose Zinc treatment, the levels of tissue Malondialdehyde (MDA) were significantly decreased, while tissue antioxidant indices were significantly increased. Meanwhile, the level of NF-κB was significantly lower compared to I/R group, while the levels of Nrf2-dependent antioxidant enzymes were significantly higher in Low-dose Zinc+I/R group. In vitro, Low-dose Zinc markedly increased Leydig cell (TM3) cell viability, and relieved testicular oxidative damage via down-regulating ROS. A total of 22 differently expressed microRNAs were screened out using microRNA microarray in rat testicular tissue caused by I/R injury, especially showing that miR-101-3p was selected as the target miRNA. Furthermore, the levels of Nrf2 and NF-κB were apparently increased/decreased in TM3 cells treated with Hypoxic/Reoxygenation (H/R) after miR-101-3p mimics/inhibitor. In addition, H/R-induced testicular oxidative damage was recovered in TM3 administrated with miR-101-3p inhibitor and si-Nrf2. Therefore, this study provided a novel insight for investigating protective effect of Zinc on testicular I/R injury by promoting antioxidation via miR-101-3p/Nrf2.