Acrylamide (ACR) is an important industrial chemical used primarily in the production of polymers and co-polymers. Acrylamide is mainly neurotoxic to experimental animals as well as humans and has also been shown to be mutagenic and carcinogenic. The present study was designed to investigate the toxicity of ACR on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and were incubated with different concentrations of ACR (0.1, 1, 10mm) for 2 hours. Cell viability by trypan blue exclusion and leakage of the enzymes such as alanine transaminase (ALT) and aspartate transaminase (AST) were determined. Reduced glutathione (GSH), glutathione S-transferase (GST) activity were also measured. A significant decrease in the cell viability was observed after exposure to 10mm ACR for 30min, while 1mm ACR caused a significant decrease in the viability after 60min. ALT leakage was parallel to the cell viability. AST leakage was significantly increased at 30min of incubation with 10mm ACR, whereas 2 hours of incubation was required for the leakage of AST from rats hepatocytes with 1mm ACR. 10mm ACR decreased significantly GSH as early as 30min, while GSH level was decreased at 60min after exposure to 1mm ACR. Also, the GST activity increased with increasing the dose of ACR. Cytochrome P450 concentration was decreased after exposure to 10mm ACR. The effect of ACR on cell viability, ALT and AST leakage, GSH and GST activity was time and dose dependent.