PII proteins are signal transduction proteins that belong to a widely distributed family involved in modulating various bacterial metabolisms. These homotrimeric proteins carry a flexible loop, known as the T-loop, whose conformation changes upon recognition of key metabolites such as ADP, ATP, and 2-oxoglutarate. PII proteins interact with various partners to primarily regulate nitrogen pathways, and in some organisms, they can also control carbon metabolism by interacting with the biotin carboxyl carrier protein (BCCP), a critical component of the acetyl-CoA carboxylase (ACC) enzyme complex, inhibiting its activity and consequently reducing fatty acid biosynthesis. In mycobacteria, only one PII protein has been identified; however, its physiological role remains unknown. In this study, we constructed a M. smegmatis deletion mutant, ∆MsPII, which exhibited growth deficiency when nitrate and nitrite were the sole nitrogen sources, leading to nitrite accumulation in the culture supernatant.