Renal ischemia/reperfusion (I/R) is a common cause of acute renal failure in many clinical settings. Our study aimed to elucidate the role of lentiviral vector-mediated KSR1 gene silencing in inflammatory factor expression and proliferation of renal tubular epithelial cells (RTECs) in a rat model of I/R injury. Male Sprague-Dawley (SD) rats were used for I/R model establishment and subject to different treatments, followed by the measurement of neurological severity score (NSS), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, 47-kDa heat-shock protein (HSP47), KSR1, and factors related to the Ras/MAPK pathway, as well as cell apoptosis. As compared with the blank group, the neurologic impairment induced by I/R in the siKSR1, U0126, and siKSR1 + U0126 groups was alleviated. Compared with the control group, the other five groups showed increased levels of TNF-α, IL-6, IL-1β, HSP47, N-ras, Raf-1, c-fos, TNF-α, IL-6, p38 MAPK, and cell apoptosis, accompanied by a declined mRNA and protein level of Bcl-2. As compared with the blank and NC groups, the siKSR1, U0126, and siKSR1 + U0126 groups showed decreased levels of TNF-α, IL-6, IL-1β, HSP47, N-ras, Raf-1, c-fos, TNF-α, IL-6, p38 MAPK, cleaved caspase-3, cleaved caspase-9, p53, and cell apoptosis, accompanied by an increased mRNA and protein level of Bcl-2. Our findings demonstrated that KSR1 gene silencing might inhibit the expression of inflammatory factors in RTECs and promote their proliferation by inactivating the Ras/MAPK pathway in the rat model of I/R injury.