Consumer interest in probiotic bifidobacteria is increasing, but industry efforts to secure high cell viability in foods is determined by these anaerobes’ sensitivity to oxidative stress. To address this limitation, we investigated genetic and physiological responses of two fully sequenced Bifidobacterium animalis subsp. lactis strains, BL-04 and DSM 10140, to hydrogen peroxide (H2O2) stress. Although the genome sequences for these strains are highly clonal, prior work showed they differ in both intrinsic and inducible H2O2 resistance. Transcriptome analysis of early stationary phase cells exposed to a sub-lethal H2O2 concentration detected significant (P2O2 stress resistance might be due to a mutation in a BL-04 gene encoding long chain fatty acid-coA ligase. To explore this possibility, membrane fatty acids were isolated and analyzed by GC-MS. Results confirmed the strains had significantly different lipid profiles; the BL-04 membrane contained higher percentages of C14:0 and C16:0, and lower percentages of C16:1n7 and C18:1n9. Alteration of the DSM 10140 membrane lipid composition using modified growth medium to more closely mimic that of BL-04 yielded cells that showed increased intrinsic resistance to lethal H2O2 challenge, but did not display an inducible H2O2 stress response. Results show deliberate stress induction or membrane lipid modification can be employed to significantly improve H2O2 resistance in B. animalis subsp. lactis strains.