Abstract The beneficial effect that melatonin has against mitochondrial dysfunctioning seems to be linked to mitophagy. Roles for melatonin have been demonstrated in promoting health and preventing disease, as well as activating the process of autophagy in general. However, no reports have been made about how the application of melatonin regulates that process when plants are exposed to oxidative stress. We investigated the influence of different concentrations of melatonin (0.0, 0.5, 5.0, 10.0, or 50.0 μ m ) on autophagy under methyl viologen ( MV )‐induced oxidative stress. Arabidopsis seedlings that were pretreated with 5 or 10 μ m melatonin underwent relatively strong induction of autophagy, as evidenced by the number of monodansylcadaverine ( MDC )‐stained autophagosomes in root samples. Pretreatment with 10 μ m melatonin also alleviated MV ‐induced photo‐oxidation damage and significantly reduced the accumulation of oxidized proteins. Those responses might have been due to the strong upregulation of genes that involved in At ATG 8‐ PE conjugation pathway, which enhanced the capacity for autophagy. Histochemical staining revealed that both and H 2 O 2 were highly accumulated upon MV exposure, although the response did not differ significantly between control and melatonin‐pretreated seedlings. By contrast, exogenous melatonin upregulated the expression of two genes for H 2 O 2 ‐scavenging enzymes, that is, At APX 1 and At CAT s . The activation of autophagy by melatonin without an alteration in ROS production may be part of a survival mechanism that is enhanced by melatonin after cellular damage. Therefore, it represents a second level of defense to remove damaged proteins when antioxidant activities are compromised.