Shigella spp. are Gram-negative bacteria that cause severe diarrheal disease, contributing significantly to morbidity and mortality worldwide. High transmissibility and increased antibiotic resistance have propelled the development of Shigella vaccine candidates; however, no FDA-approved vaccine exists to date. We have collaborated with Walter Reed Army Institute of Research (WRAIR) to improve their live-attenuated vaccine candidates, whose only drawback during clinical trials was febrile symptoms experienced in a few individuals. In this study, we sought to resolve those unwanted side effects by detoxifying the lipid A moiety (also known as endotoxin) that is thought to contribute to the febrile symptoms observed. To do so, we employed bacterial enzymatic combinatorial chemistry (BECC), whereby lipid A modification enzymes were ectopically expressed to induce structural alterations known to dampen lipid A signaling capacity. The enzymes LpxE (phosphatase) and PagL (deacylase) were expressed in both virulent and attenuated vaccine strains of Shigella to modify the bis-phosphorylated hexa-acylated lipid A structure ordinarily present in the Shigella outer membrane. The expected modifications were confirmed using mass spectrometric and gas chromatographic analyses when the enzymes were expressed individually or in combination (both LpxE and PagL) using a construct we refer to as “Dual”. These enzyme/enzyme combinations were subsequently integrated into the chromosome using Tn7 transposition to avoid the possibility of plasmid loss during production. The impact of the induced lipid A structural alterations on innate immune signaling was assessed by stimulation of NF-?B reporter cell lines and human PBMCs with the extracted lipopolysaccharide (LPS). Additionally, in vivo reactogenicity of the LPS in a murine acute endotoxemia model was assessed. We found that dephosphorylation, but not deacylation, of lipid A, was a powerful tool to reduce LPS-mediated signaling in live-attenuated Shigella vaccines, resulting in reduced toxicity of Shigella LPS in vivo. Additionally, we found that dephosphorylation of the lipid A moiety did not impair invasion of colonic epithelia or immunogenicity in a mouse pulmonary model. Overall, this study generated Shigella vaccine candidates with reduced endotoxicity, which upon oral ingestion in humans, will ultimately have reduced reactogenicity.