Aims: To elucidate and characterize the metabolic putrescine synthesis pathway from agmatine by Lactobacillus hilgardii X1B. Methods and Results: The putrescine formation from agmatine by resting cells (the normal physiological state in wine) of lactic acid bacteria isolated from wine has been determined for the first time. Agmatine deiminase and N-carbamoylputrescine hydrolase enzymes, determined by HPLC and LC-Ion Trap Mass Spectrometry, carried out the putrescine synthesis from agmatine. The influence of pH, temperature, organic acids, amino acids, sugars and ethanol on the putrescine formation in wine was determined. Conclusions: Resting cells of Lact. hilgardii X1B produce putrescine in wine. The putrescine production was carried out from agmatine through the agmatine deiminase system. Significance and Impact of the Study: These results have significance from two points of view, wine quality and toxicological and microbiological aspects, taking account that putrescine, which origin is still controversial, is quantitatively the main biogenic amine found in wine.