Mycobacterial membrane proteins play a pivotal role in the bacterial invasion of host cells; however, the precise mechanisms underlying certain membrane proteins remain elusive. (Ms) is a hemolysin III family protein that is homologous to (Mtb) , but it has an unclear function in growth. To address this issue, we utilized the CRISPR/Cas9 gene editor to construct Δ strains and combined RNA transcription and LC-MS/MS protein profiling to determine the functional role of in Ms growth. The correlative analysis showed that the deletion of inhibits ABC transporters in the cytomembrane and inhibits the biosynthesis of amino acids in the cell wall. Corresponding to these results, we confirmed that MSMEG5257 localizes in the cytomembrane via subcellular fractionation and also plays a role in facilitating the transport of iron ions in environments with low iron levels. Our data provide insights that plays a role in maintaining Ms metabolic homeostasis, and the deletion of significantly impacts the growth rate of Ms. Furthermore, , a promising drug target, offers a direction for the development of novel therapeutic strategies against mycobacterial diseases.