Background: An advanced age associated pathology is decreasing cognitive ability and impeding antioxidant system integrity. Adverse drug reactions have prompted the need for complementary and alternative medicine dietary therapy. Buffalo milk is reported to have high levels of various nutrients which makes it an ideal candidate for complementary dietary therapy. However, its effects on oxidation, fatigue, learning and memory potential remains to be explored. Results: Compared with cow milk, buffalo milk showed better anti-fatigue effect evidenced by improved force swimming time, elevated lactate dehydrogenase, hepatic glycogen, and decline of blood urea nitrogen (P<0.05), especially in young female mice. Pasteurized buffalo milk (PBM) and high-temperature treatment sterilized buffalo milk (HTBM) both rescued the serum and liver anti-oxidative enzymes impaired by D-galactose treatment. However, PBM supplement showed higher liver superoxide dismutase (P<0.05) and glutathione peroxidase level (P<0.05) than HTBM in D-galactose induced aging mice. PBM and HTBM both enhanced the priority and discrimination indexes for object recognition and locations tests in D-galactose induced aging mice model. Supplement of PBM or HTBM successfully restored brain superoxide dismutase, glutathione peroxidase levels, and brain protein content in D-galactose induced aging mice (P<0.05). Meanwhile, the elevated malondialdehyde levels were also decreased (P<0.05) by either PBM or HTBM treatment. Conclusion: Our present study established that buffalo milk, as a natural dairy product, could alleviate fatigue in young mice, and rescue oxidative stress and promote learning and memory in aging mice. Pasteurized buffalo milk showed more favorable effects on aging mice when compared with high-temperature treatment sterilized buffalo milk.