Abstract: Objective To describe the protein secretion activity of M.chelonae, and to provide the research basis for its pathogenicity and detection methods.Methods With weak cation exchange protein chip (WCX2), Middlebrood 7H9 culture filtrate proteins for 7 days’ culture and Middlebrook 7H9 culture proteins of 11 M.chelonae clinical isolates and ATCC19977 reference strain, were detected by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), the M.chelonae, culture filtrate proteins were analyzed descriptively.Results 49－101 kinds of differential proteins were detected in 11 M.chelonae Middlebrook 7H9 culture filtrate for 7 days’ culture, the relative molecular mass ranged from 1101－3953, and the abundance level in relative strength were 84－7238. Thirty-two kinds of differential proteins were detected in 11 M.chelonae, the molecular mass ranged from 1108-3953, and the abundance level in relative strength were 98－7231.Conclusion A variety of small proteins were secreted by M.chelonae in the process of growth in vitro, while there characteristics and effects needs further research.