Aims To evaluate the effect of cultivation (anaerobiosis vs respiration) and Tween 80 supplementation on the production of metabolites and on the composition of membrane fatty acids (FAs) in Lactobacillus casei N87. Methods and Results Anaerobic and respiratory growth, with or without Tween 80 supplementation, was carried out in a chemically defined medium. Production of biomass, organic acids, volatile organic compounds (VOCs), consumption of amino acids and changes in membrane FAs were investigated. Respiration altered the central metabolism rerouting pyruvate away from lactate accumulation, while Tween 80 had a minor effect on metabolic pathways. VOCs were mainly affected by growth conditions and significant amounts of diacetyl were produced by respiratory cultures. Respiration increased desaturation of membrane lipids and Tween 80 improved the production of essential polyunsaturated FAs. Palmitic acid decreased in Tween-supplemented aerated cultures. Conclusions Combination of Tween 80 and respiratory growth promoted production of biomass and aroma compounds and affected the composition of membrane FAs in Lact. casei N87. Significance and Impact of the Study Respiration might be exploited in Lact. casei as a natural strategy for the enhanced production of aroma compounds.