Present study was conducted to investigate the acute and sub-acute toxic effect of diplodiatoxin with special reference to biochemical membrane bound enzymes like aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and RBC acetylcholinesterase (AChE) in male and female rats. For acute study, rats were treated with a single oral dose of 5.7 mg/kg of diplodiatoxin, whereas for sub-acute study, the rats received 0.27 mg/kg/day for 21 days. Acute and sub-acute diplodiatoxin treatment caused loss in body weight and feed intake along with symptoms including irritation, dullness, tremors and convulsions. Diplodiatoxin caused a significant increase in serum ASAT and ALAT and a decrease in activity in the liver in both acute and sub-acute studies. This compound also significantly inhibited RBC AChE. Sexual dimorphism was observed when male rats were compared with female rats (p < 0.05). The enzyme alterations observed in the affected enzymes recovered to the normal levels by day 7 post treatment (withdrawal study) in both acute and sub-acute treated rats. A negative correlation was observed with regard to these enzymes when serum was compared with liver. These enzyme profiles show increases in serum with parallel decrease in liver, indicating necrosis of liver. These results suggest that diplodiatoxin has potential to affect hepatic end-points.