SummaryLipid A anchors the lipopolysaccharide (LPS) to the outer membrane and is usually composed of a hexa‐acylated diglucosamine backbone. Burkholderia cenocepacia, an opportunistic pathogen, produces a mixture of tetra‐ and penta‐acylated lipid A. “Late” acyltransferases add secondary acyl chains to lipid A after the incorporation of four primary acyl chains to the diglucosamine backbone. Here, we report that B. cenocepacia has only one late acyltransferase, LpxL (BCAL0508), which adds a myristoyl chain to the 2′ position of lipid A resulting in penta‐acylated lipid A. We also identified PagL (BCAL0788), which acts as an outer membrane lipase by removing the primary β‐hydroxymyristate (3‐OH‐C14:0) chain at the 3 position, leading to tetra‐acylated lipid A. Unlike PagL, LpxL depletion caused reduced cell growth and defects in cell morphology, both of which were suppressed by overexpressing the LPS flippase MsbA (BCAL2408), suggesting that lipid A molecules lacking the fifth acyl chain contributed by LpxL are not good substrates for the flippase. We also show that intracellular B. cenocepacia within macrophages produced more penta‐acylated lipid A, suggesting lipid A penta‐acylation in B. cenocepacia is required not only for bacterial growth and morphology but also for adaptation to intracellular lifestyle.