Swamp buffaloes accounted for the majority of China buffaloes, the yield of milk is high in quality but low in quantity. The lactating mechanism is largely unknown. To fill the gap, we analyzed the proteome of buffalo mammary gland in lactating and non-lactating periods using proteomics based on isobaric tags. Mammary gland tissues from both lactating and non-lactating (in dry period) buffaloes were collected from a local slaughterhouse in Nanning, Guangxi province, China. Animals of similar weight (500 kg) and age of (10 years) were used for the sample collection.