[Objective] To understand the role of acetylation modification on the activity of Ku protein.[Methods] Acetylated Ku protein and its non-acetylated mutant were expressed and purified using M smegmatis expression system, and then their biochemical activities were compared. The effect of oxidative stress and acidic environment on Ku protein acetylation level were analyzed in M. smegmatis. [Results] Ku protein over-expression M. smegmatis strain grew slower than the control strain transformed with the empty plasmid. Acetylated Ku protein had lower DNA repair activity and DNA binding activity than the non-acetylated Ku. Quantity of Ku protein in M. smegmatis cells under oxidative and acidic stress decreased, whereas there was subtle change of acetylated Ku protein. [Conclusion] Acetylation modification can regulate the DNA binding activity of Ku protein, thus regulate the activity of Non-homologous end joining system. The increase of acetylation level of Ku protein is the response of mycobacteria against the adverse growth environment.