Abstract Background Pyruvate kinase (Pyk) catalyzes the generation of pyruvate and ATP in glycolysis and functions as a key switch in the regulation of carbon flux distribution. Both the substrates and products of Pyk are involved in the tricarboxylic acid cycle, anaplerosis and energy anabolism, which places Pyk at a primary metabolic intersection. Pyks are highly conserved in most bacteria and lower eukaryotes. Corynebacterium glutamicum is an industrial workhorse for the production of various amino acids and organic acids. Although C. glutamicum was assumed to possess only one Pyk ( pyk1 , NCgl2008), NCgl2809 was annotated as a pyruvate kinase with an unknown role. Results Here, we identified that NCgl2809 was a novel pyruvate kinase ( pyk2 ) in C. glutamicum . Complementation of the WTΔ pyk1 Δ pyk2 strain with the pyk2 gene restored its growth on d -ribose, which demonstrated that Pyk2 could substitute for Pyk1 in vivo . Pyk2 was co-dependent on Mn 2+ and K + and had a higher affinity for ADP than phosphoenolpyruvate (PEP). The catalytic activity of Pyk2 was allosterically regulated by fructose 1,6-bisphosphate (FBP) activation and ATP inhibition. Furthermore, pyk2 and ldhA , which encodes l -lactate dehydrogenase, were co - transcribed as a bicistronic mRNA under aerobic conditions and pyk2 deficiency had a slight effect on the intracellular activity of Pyk. However, the mRNA level of pyk2 in the wild-type strain under oxygen deprivation was 14.24-fold higher than that under aerobic conditions. Under oxygen deprivation, pyk1 or pyk2 deficiency decreased the generation of lactic acid, and the overexpression of either pyk1 or pyk2 increased the production of lactic acid as the activity of Pyk increased. Fed-batch fermentation of the pyk2 -overexpressing WTΔ pyk1 strain produced 60.27 ± 1.40 g/L of lactic acid, which was a 47% increase compared to the parent strain under oxygen deprivation. Conclusions Pyk 2 functioned as a pyruvate kinase and contributed to the increased level of Pyk activity under oxygen deprivation.