This study was performed to determine the median lethal concentration (96-h LC50) of penoxsulam (PNX) in Nile tilapia fingerlings and to investigate the protective effect of vitamin E (Vit E) against biochemical and immunological alterations induced by two sub-lethal concentrations of PNX (1/10 and 1/20 of 96-h LC50). The 96-h LC50 of PNX was found to be 3.1 mg/l. Fish was exposed to sub-lethal concentrations of PNX for 45 days. The fish exposed to Vit E + PNX were fed with Vit E-supplemented diet (700 mg/kg diet) daily for 10 days then exposed to the same concentrations of PNX and fed on Vit E-supplemented diet for 45 days. The results showed that PNX exposure caused significant concentration-dependent increases in serum alanine transaminase, aspartate transaminase, creatinine, malondialdehyde and nitric oxide and significant decreases in serum total protein, albumin, reduced glutathione, glutathione peroxidase, superoxide dismutase and lysozyme activity. Furthermore, PNX significantly up-regulated the expression of immune-related genes in the head kidney in a concentration-dependent manner when compared with the control group. However, Vit E supplementation in the diet alleviated PNX-induced toxic effects. These results concluded that Vit E could be effective in the protection of fish against PNX toxicity.