This work deals with the production of reuterin by Lactobacillus reuteri PRO 137 in a two-step batch fermentation process. First, L. reuteri cells were grown anaerobically in a glucose-containing media, and in a second stage reuterin was produced by the starved resting cells in glycerol-containing media. In order to maximize biomass production, the influence of temperature (31–43 °C) and pH (5.0–6.5) was evaluated. A simple model has been proposed to describe the microbial growth and substrate consumption. The values of the kinetic constant for growth rate, yield of biomass on substrate and maximum biomass concentration indicate that the optimum operating conditions were 40 °C and pH 5.5. Biomass produced in those growth conditions was harvested, washed and resuspended in a medium containing glycerol as the only carbon source for reuterin production by resting cells. Reuterin production was evaluated for several biomass (2 and 5 CDW g/L) and glycerol (150–300 mM) concentrations. A model describing the kinetics of the production process has been proposed. The model allows to describe simultaneously the concentrations of viable cells, glycerol, reuterin and 1,3-propanediol. A multi-response nonlinear parameter estimation procedure using the Marquardt algorithm and numerical integration of the differential equations by Runge-Kutta method was used to obtain the values of the fitting parameters.