Some microorganisms accumulate glucosylglycerate (GG) during growth under nitrogen deprivation. However, the molecular mechanisms underlying the role of GG and the regulation of its levels in the nitrogen stress response are elusive. Since GG is required for biosynthesis of mycobacterial methylglucose lipopolysaccharides (MGLP) we examined the molecular mechanisms linking replenishment of assimilable nitrogen to nitrogen-starved M. hassiacum with depletion of GG accumulated during nitrogen deficiency. To probe the involvement of a newly identified glycoside hydrolase in GG depletion, we produced the mycobacterial enzyme recombinantly and confirmed the specific hydrolysis of GG (GG hydrolase, GgH) in vitro.We have also observed a pronounced up-regulation of GgH mRNA in response to the nitrogen shock, which positively correlates with GG depletion in vivo and growth stimulation, implicating GgH in the recovery process. Since GgH orthologs seem to be absent from most slowly-growing mycobacteria including M. tuberculosis, the disclosure of the GgH function allows reconfiguration of the MGLP pathway in rapidly-growing species and accommodation of this possible regulatory step. This new link between GG metabolism, MGLP biosynthesis and recovery from nitrogen stress furthers our knowledge on the mycobacterial strategies to endure a frequent stress faced in some environments and during long-term infection.