Protein phosphorylation plays versatile and important roles in the pathogen and host physiology in Mycobacterium tuberculosis, which holding promise as new targets for anti-tuberculosis drugs. Mycolicibacterium smegmatis was used as a model organism to study the protein phosphorylation at different growth phases because of its close association with M.tuberculosis. We identified 573 phosphorylated peptides and 933 phosphorylation sites from 385 proteins in samples from both logarithmic and stationary phase of M.smegmatis. The number of identified phosphorylation sites is 4.2 times higher than the largest phosphoproteome discovered to date, establishing a comprehensive dataset of phosphorylated proteins in M.smegmatis. By comparing the significantly differentially expressed phosphorylated proteins between the logarithmic phase and the stationary phase, we discovered 68 upregulated proteins involved in cell elongation and division pathways and 69 downregulated proteins involved in signaling pathways such as the tricarboxylic acid cycle. Differentially expressed proteins were found to regulate important cellular cycle events such as cell growth and proliferation, providing proteome level support for the elucidation of phosphorylation functions in both M. smegmatis and M. tuberculosis.