Lactulose has profound health benefits by way of increasing bifidobacterial flora in the intestine of infants thereby protecting them against enteric infection, constipation and systemic encephalopathy. In the present study to assess the sub chronic toxicity of lactulose syrup, the rats were fed on a basal feed supplemented with lactulose syrup at 0.5, 1.0, 2.0 and 5.0% for a period of 21 weeks. Monitoring of food consumption, gain in body weight and physical observations did not reveal any treatment-related toxicity in any of the group of rats. Terminal autopsy also did not reveal any signs of toxicity. Further, no significant alterations in relative organ weight, serum biochemistry and urinalysis were observed up to 1% lactulose supplementation level. The results suggest that supplementation of lactulose in the diet does not produce any toxicity at the doses tested.