Burkholderia pseudomallei, the etiological agent of melioidosis, is a facultative intracellular pathogen. As B. pseudomallei is a gram-negative bacterium, its outer membrane contains lipopolysaccharide (LPS) molecules, which have been shown to have low-level immunological activities in vitro. In this study, the biological activities of B. pseudomallei LPS were compared to those of Burkholderia thailandensis LPS, and it was found that both murine and human macrophages produced levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-10 in response to B. pseudomallei LPS that were lower than those in response to B. thailandensis LPS in vitro. In order to elucidate the molecular mechanisms underlying the low-level immunological activities of B. pseudomallei LPS, its lipid A moiety was characterized using mass spectrometry. The major lipid A species identified in B. pseudomallei consists of a biphosphorylated disaccharide backbone, which is modified with 4-amino-4-deoxy-arabinose (Ara4N) at both phosphates and penta-acylated with fatty acids (FA) C(14:0)(3-OH), C(16:0)(3-OH), and either C(14:0) or C(14:0)(2-OH). In contrast, the major lipid A species identified in B. thailandensis was a mixture of tetra- and penta-acylated structures with differing amounts of Ara4N and FA C(14:0)(3-OH). Lipid A species acylated with FA C(14:0)(2-OH) were unique to B. pseudomallei and not found in B. thailandensis. Our data thus indicate that B. pseudomallei synthesizes lipid A species with long-chain FA C(14:0)(2-OH) and Ara4N-modified phosphate groups, allowing it to evade innate immune recognition.