To explore the mechanism of renal toxicity induced by florfenicol (FFC), 120 chicks were randomly divided into 6 groups, 20 in each group. Except for the control group, different doses of FFC (0.15 g/L, 0.3 g/L, 0.6 g/L, 1.2 g/L, and 1.8 g/L) were added to drinking water in the other 5 groups. Five days later, blood was collected from the vein under the wing, and the complete kidneys were obtained as soon as possible, then tested the experimental indicators. The results showed that compared with control group, all doses of FFC significantly reduced the average weight gain of chicks (P < 0.05 or P < 0.01). Except for the 0.15 g/L FFC group, kidney index of chicks in the other doses of FFC groups were significantly increased (P < 0.05 or P < 0.01). The kidney tissues in all FFC groups showed obvious damage, deformities, cell atrophy, and cell gap enlargement. In addition, all doses of FFC significantly increased the contents of uric acid (UA), blood urea nitrogen (BUN), creatinine (CRE) in serum, and malondialdehyde (MDA) in renal tissue (P < 0.05 or P < 0.01), but significantly reduced the levels of glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) in renal tissue (P < 0.05 or P < 0.01). FFC significantly inhibited the mRNA and protein expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase-1 (NQO-1), and increased the mRNA and protein expression levels of p53, Caspase-3, and Caspase-6 (P < 0.05 or P < 0.01). The apoptotic rate of renal cells in all doses of FFC groups increased significantly (P < 0.05 or P < 0.01). It was concluded that FFC had a certain degree of nephrotoxicity, and with the increase of FFC concentration, the kidney injury of chicks became more and more serious. FFC promoted oxidative stress response in kidney of chicks by inhibiting the expression of related factors in Nrf2-ARE pathway. Moreover, the expression of pro-apoptotic factors was upregulated to improve the apoptosis rate of renal cells, which resulted in excessive apoptosis of renal cells and seriously affected the kidney function of chicks.