Mycobacterium tuberculosis Rv2140c is a function unknown conserved phosphatidylethanolamine-binding protein (PEBP), homologous to Raf kinase inhibitor protein (RKIP) in human beings. To delineate its function, we heterologously expressed Rv2140c in a non-pathogenic M. smegmatis. Quantitative phosphoproteomic analysis between two recombinant strains Ms_Rv2140c and Ms_vec revealed that Rv2140c differentially regulate 425 phosphorylated sites representing 282 proteins. Gene ontology GO, and a cluster of orthologous groups COG analyses showed that regulated phosphoproteins by Rv2140c were mainly associated with metabolism and cellular processes. Rv2140c significantly repressed phosphoproteins involved in signaling, including serine/threonine-protein kinases and two-component system, and the arabinogalactan biosynthesis pathway phosphoproteins were markedly up-regulated, suggesting a role of Rv2140c in modulating cell wall. Consistent with phosphoproteomic data, Rv2140c altered some phenotypic properties of M. smegmatis such as colony morphology, cell wall permeability, survival in acidic conditions, and active lactose transport. In summary, we firstly demonstrated the role of PEBP protein Rv2140c, especially in phosphorylation of mycobacterial arabinogalactan biosynthesis proteins.