3-Hydroxypropionaldehyde (3-HPA) is a potential valuable chemical and new broad-spectrum antimicrobial substance. In order to improve the conversion of 3-HPA/glycerol, our work studied the two-step process from glycerol to 3-HPA, and investigated the influence of cell harvest time, glycerol concentration, biomass concentration, pH and temperature on the production of 3-HPA by Lactobacillus reuteri CG001, respectively. The results showed that molar conversion yield of 3-HPA/glycerol reached 97.9% under the condition that 200 mmol.L(-1) glycerol was converted by 25.3 g.L(-1) resting cell for 1 h at 30 degrees C. The cells could not be reused directly because the L. reuteri almost lost its bioconversion activity completely, but the ability of glycerol conversion could gradually recover if the fresh medium was added to the deactivated cell for 4 h.