Lipid A, the hydrophobic anchor of lipopolysaccharide, is an essential component in the outer membrane of most gram-negative bacteria. It is recognized by the TLR4/MD2 receptor of the innate immune system, which triggers an inflammatory response accompanied by massive overproduction of cytokines and leads to gram-negative septic shock. Human pathogen Klebsiella pneumoniae, which may synthesize two lipid A species, differs by the length of the secondary acyl chain. In this study, we identified two genes encoding the putative late acyltransferases of lipid A biosynthesis pathway in K. pneumoniae. Based on the sequence alignment, proteins YP_002239312.1 encoded by KPK3489 and YP_002239899.1 encoded by KPK4096 are homologous to E. coli LpxL, which were designated as LpxL1 and LpxL2, respectively. Functions of the two acyltransferases were confirmed by overexpressing the genes in E. coli, isolating lipid A and analyzing their structures using an ESI/MS. Like E. coli LpxL, K. pneumoniae LpxL1 transfers a C12:0 secondary acyl chain to the 2'-position of lipid A, while K. pneumoniae LpxL2 transfers a C14:0 secondary acyl chain to the 2'-position primary acyl chain of lipid A. These two acyltransferases might play important roles in the biosynthesis of lipid A and the innate immune system recognition in K. pneumoniae.