Shigella flexneri is a major cause of gastro-enteric disease in the developing world, affecting mostly children under the age of 5. This bacterial pathogen biosynthesises a plethora of complex lipopolysaccharides (LPS) on its cell surface that facilitate host cell-surface adhesion and can modulate immune responses. S. flexneri expresses LPS with two distinct O antigen (Oag) polysaccharide chain lengths, referred to as short-Oag (S-Oag; 10-17 tetrasaccharide repeat units) and very long-Oag (VL-Oag; >90 tetrasaccharide repeat units). However, how the Oag length of LPS influences the interaction with hydrophobic host molecules, such as fatty acids is poorly understood. In this study, S. flexneri was found to experience perturbed growth in the presence of antimicrobial host fatty acids. Notably, the degree of susceptibility was directly linked to the length of the LPS Oag expressed, with strains expressing VL-Oag exhibiting greater resistance compared to strains devoid of Oag or expressing exclusively S-Oag. Consistently, fatty acid acquisition analyses via fluorescent labelling techniques or cellular fatty acid profiling illustrated that the S. flexneri Oag length-dependent relationship directly affects the ability of this pathogen to acquire fatty acids from its environment. We also defined that the reduced uptake of antimicrobial fatty acids in the VL-Oag expressing S. flexneri protected the bacteria from dramatic, and potentially detrimental, lipidomic shifts. Interestingly, the exposure to antimicrobial fatty acids did not affect Oag biosynthesis. Collectively, this work revealed a new role for LPS in lipid homeostasis in a critical human pathogen. By interrogating this link between distinct components of the microbial envelope, we have provided clinically significant insights into optimising treatment of this pathogen, as host fatty acids are known to influence antibiotic efficacy at the host-pathogen interface.