Abstract: Objective To test the acute and chronic toxicity of topical application of 0.5% podophyllotoxin-loaded nanostructured lipid carriers (POD-NLC) to the vaginal mucosa. Methods Twelve New Zealand rabbits were randomized into 3 groups and subjected to daily topical applications of normal saline (control group), 0.5% podophyllotoxin tincture (POD-T) or 0.5% POD-NLC on the vaginal mucosa for 10 consecutive days, and the pathological changes in the mucosa were graded using the Eckstein scoring system. The acute toxicity of POD-NLC was tested in 20 SD female rats, which received intravaginal administration of POD-NLC or vehicle for 3 times within 24 h; After 14 days of continuous observation, the rats were dissected for calculating the viscera coefficient. For testing the chronic toxicity of POD-NLC, 80 SD female rats were randomized into 4 groups and subjected to daily intravaginal administration of the vehicle or POD-NLC at low, moderate or high doses for 13 consecutive weeks. The rats were weighed once a week and at the end of the experiment, 2/3 of the rats from each group were sacrificed to collect blood samples, calculate the viscera coefficient, and examine the pathological changes in the liver. The remaining 1/3 rats were observed for another 2 weeks without further drug treatment and the same examinations were performed. Results In the rabbits, 0.5% POD-NLC elicited only mild irritation while POD-T caused moderate irritation of the vaginal mucosa. In the acute toxicity test, the organ coefficients were comparable between the rats treated with the vehicle and POD-NLC (P>0.05). Long-term intravaginal administration of POD-NLC did not produce significant changes in the behavior, activity, body weight, blood biochemical profiles or organ coefficient as compared with the vehicle control group (P>0.05). Conclusions Intravaginal administration of 0.5% POD-NLC causes very mild irritation without obvious acute or chronic toxicity to the vaginal mucosa in rabbits and rats.