Environmental pH can be an important cue for symbiotic bacteria as they colonize their eukaryotic hosts. Using the model mutualism between the marine bacterium Vibrio fischeri and the Hawaiian bobtail squid, we characterized the bacterial transcriptional response to acidic pH experienced during the shift from planktonic to host‐associated lifestyles. We found several genes involved in outer membrane structure were differentially expressed based on pH, indicating alterations in membrane physiology as V. fischeri initiates its symbiotic program. Exposure to host‐like pH increased the resistance of V. fischeri to the cationic antimicrobial peptide polymixin B, which resembles antibacterial molecules that are produced by the squid to select V. fischeri from the ocean microbiota. Using a forward genetic screen, we identified a homolog of eptA, a predicted phosphoethanolamine transferase, as critical for antimicrobial defense. We used MALDI‐MS to verify eptA as an ethanolamine transferase for the lipid‐A portion of V. fischeri lipopolysaccharide. We then used a DNA pulldown approach to discover that eptA transcription is activated by the global regulator H‐NS. Finally, we revealed that eptA promotes successful squid colonization by V. fischeri, supporting its potential role in initiation of this highly specific symbiosis.