This study reported metabolic profiles of three representative strains from Lactobacillus species, and explored their metabolic response to visible light exposure. We utilized strains from three Lactobacillus species, Lactobacillus acidophilus, Lactobacillus fermentum and Lactobacillus delbrueckii as our model bacteria and applied mass spectrometry base targeted metabolomics to specifically investigate 221 metabolites within multiple metabolic pathways. Similar and diverse metabolome from three tested strains were discovered. Furthermore, all three Lactobacillus strains demonstrated different metabolic profiles in comparison between light expose verse control. In all three strains, 12 metabolites were detected to have significant differences (p-value < 0.01) in light exposure culture compared to the control samples (culture grown without light exposure). Principal components analysis using these significantly changed metabolites clearly separated the exposure and control groups in all three studied Lactobacillus strains. Additionally, metabolic pathway impact analysis indicated that several commonly impacted pathways can be observed.