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Autophagy is a conserved cellular process that functions in the maintenance of physiological and metabolic balance. It has previously been demonstrated to improve plant tolerance to abiotic stress. Numerous autophagy–related genes (ATGs) that regulate abiotic stress have been identified, but there have been few functional studies showing how ATGs confer cold stress tolerance. The cold transcriptome data of the crown buds that experienced overwintering of the alfalfa (<i>Medicago sativa</i> L.) showed that <i>MsATG13</i> is upregulated in response to cold stress. In the present study, we found that <i>MsATG13</i> transgenic tobacco enhanced cold tolerance compared to wild–type (WT) plants. Transmission electron microscopy demonstrated that transgenic tobacco overexpressing <i>MsATG13</i> formed more autophagosomes than WT plants in response to cold stress conditions. The transgenic tobacco increased autophagy levels due to upregulation of other ATGs that were necessary for autophagosome production under cold stress conditions. <i>MsATG13</i> transgenic tobacco also increased the proline contents and antioxidant enzyme activities, enhancing the antioxidant defense capabilities under cold stress conditions. Furthermore, <i>MsATG13</i> overexpression decreased levels of superoxide anion radicals and hydrogen peroxide under cold stress conditions. These findings demonstrate the role of <i>MsATG13</i> in enhancing plant cold tolerance through modulation of autophagy and antioxidant levels.