Kidney ischemia reperfusion (IR) contributes to the development of acute kidney injury. The hypoxic conditions in ischemic damage lead to oxidative stress and apoptotic cell death. We investigated the effects of vitamin D3 (Vit D) and erythropoietin (EPO) on microRNA-21(miR-21) expression in renal IR. Wistar rats were divided into five groups including the control, vehicle + IR, Vit D + IR, EPO + IR, and Vit D + EPO + IR groups. The animals were unilaterally nephrectomized and subjected to 45 min of renal pedicle occlusion followed by 24 h reperfusion. Vitamin D3 and EPO were administered prior to ischemia. After 24 h reperfusion, the kidney samples were collected for the detection of miR-21, heat shock protein 70 (hsp70) and caspase-3 expression levels. Kidney IR significantly increased the expression of miR-21, hsp70 and capase-3 and blood urea nitrogen (BUN)-Cr levels. Treatment with vitamin D3 and EPO significantly decreased the BUN-Cr levels and hsp70 and caspase-3 expression. Also, the co-administration of two drugs significantly increased miR-21 expression. It seems that vitamin D3 or EPO administration could protect the kidney against IR injury. However, vitamin D3 and EPO co-treatment was the most effective compared with the other treatment groups.