Effects of selenoproteins on many renal diseases have been reported. However, their role in renal ischemia-reperfusion (I/R) injury is unclear. The present study was performed to investigate the impact of ebselen and renal I/R injury on the expression of selenoproteins. Sprague-Dawley rats were pretreated with or without ebselen (10 mg/kg) through a daily single oral administration from 3 days before renal I/R surgery. RT-qPCR (real-time quantitative PCR) was performed to determine the mRNA expression of 25 selenoprotein genes in the renal tissues. The expression levels of two selenoproteins, including GPX3 (glutathione peroxidase 3) and DIO1 (iodothyronine deiodinase 1), were evaluated by Western blot or/and IHF (immunohistofluorescence) assays. Furthermore, renal function, renal damage, oxidative stress, and apoptosis were assessed. The results showed that in renal I/R injury, the mRNA levels of 15 selenoprotein genes (GPX1, GPX3, GPX4, DIO1, DIO2, TXNRD2, TXNRD3, SEPHS2, MSRB1, SELENOF, SELENOK, SELENOO, SELENOP, SELENOS, and SELENOT) were decreased, whereas those of eight selenoprotein genes (GPX2, GPX6, DIO3, TXNRD1, SELENOH, SELENOM, SELENOV, and SELENOW) were increased. I/R also induced a reduction in the expression levels of GPX3 and DIO1 proteins. In addition, our results indicated that ebselen reversed the changes in those selenoprotein genes, excluding SELENOH, SELENOM, SELENOP, and SELENOT, in renal I/R injury and alleviated I/R-induced renal dysfunction, tissue damage, oxidative stress, and apoptosis. To our knowledge, this is the first study to investigate the changes of 25 mammalian selenoprotein genes in renal I/R injury kidneys. The present study also provided more evidence for the roles of ebselen against renal I/R injury.