Aims: This study was conducted to understand the influences of fermentation factors in NADH recycling and mechanisms of 1,3-propanediol (1,3-PDO) production in Lactobacillus panis PM1. Methods and Results: We conducted metabolite analyses, qRT-PCR of the glycerol reductive pathway [glycerol dehydratase (DhaB) and 1,3-PDO dehydrogenase (DhaT)] and DhaT activity assays at different pH, temperature and initial glycerol concentrations. The supplementation of 150mmoll(-1) glycerol caused a shift in NADH flux from ethanol to 1,3-PDO production, whereas 300moll(-1) glycerol negatively affected the regeneration of NAD(+) via 1,3-PDO production. This retardation decreased transcription levels and specific activities of DhaT. The decreased DhaT activity eventually caused the shutdown of 1,3-PDO production. Temperature and pH did not significantly affect the specific activity of DhaT, whereas expression of genes for DhaB and DhaT was activated under acidic conditions. Moreover, fresh glucose addition after its depletion could not restart the glycerol reduction, but increased ethanol production. Conclusions: Those environmental factors affect 1,3-PDO production in different ways through changing the expression level of enzymes and shifting the NAD(+) regeneration pathways. Significance and Impact of the Study: Our findings elucidated a key element to optimize 1,3-PDO production by Lact.panis PM1, which potentially improves 1,3-PDO manufacturing efficiencies.