Lactobacillus reuteri grown in MRS broth containing 20 mM glycerol exhibits 3.7-fold up-regulation of 3-hydroxypropionic acid (3-HP) pathway genes during the stationary phase. Concomitantly, the resting cells prepared from stationary phase show enhancement in bio-conversion of glycerol, and the maximum specific productivity (q (p)) is found to be 0.17 g 3-HP per g CDW per hour. The regulatory elements such as catabolite repression site in the up-stream of 3-HP pathway genes are presumed for the augmentation of glycerol bio-conversion selectively in stationary phase. However, in the repression mutant, the maximum q (p) of 3-HP persisted in the stationary phase-derived resting cells indicating the role of further regulatory features. In the production stage, the external 3-HP concentration of 35 mM inhibits 3-HP synthesis. In addition, it has also moderated 1,3-propanediol formation, as it is a redox bio-catalysis involving NAD(+)/NADH ratio of 6.5. Repeated batch bio-transformation has been used to overcome product inhibition, and the total yield (Ypx) of 3-HP from the stationary phase-derived biomass is 3.3 times higher than that from the non-repeated mode. With the use of appropriate gene expression condition and repeated transfer of biomass, 3-HP produced in this study can be used for low-volume, high-value applications.