The anti-oxidant of bamboo leaves (AOB) has recently been certificated as a novel kind of natural anti-oxidant by the Ministry of Health of the People's Republic of China, and has been used in various food systems. Here, AOB was subjected to a series of acute and subchronic toxicological tests to evaluate its safety. It was examined to evaluate acute oral toxicity by using Kun-Ming mice and Sprague-Dawley rats, and its mutagenic potential assessed by reverse mutation test using Salmonella typhimurium, bone marrow cell micronucleus test using Kun-Ming mice, and sperm abnormality test using Kun-Ming mice. In addition, a 90-day oral toxicity study using Sprague-Dawley rats was conducted to evaluate subchronic toxicology. The results showed that the maximum tolerated dose (MTD) of AOB was >10 g/kg body weight in both rats and in mice, which can be regarded as virtually non-toxic. No mutagenicity evidence was detected in any of the three mutagenic tests. Administration at levels of 1.43, 2.87 and 4.30 g/kg per day to the rats for 90 days did not induce significant hematological, clinic, chemical and histopathological changes, and suggested a no-observed-adverse-effect level (NOAEL) of 4.30 g/kg per day. These results indicate that AOB can be generally regarded as safe for use as a food additive.