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Melatonin (N-acetyl-5-methoxytryptamine) is a tryptophan-derived signal with important physiological roles in mammals. Although the presence of melatonin in plants may be universal, its endogenous function in plant tissues is unknown. On the basis of its structural similarity to indole-3-acetic acid, recent studies mainly focusing on root growth in several plant species have suggested a potential auxin-like activity of melatonin. However, direct evidence about the mechanisms of action of this regulator is lacking. In this work, we used Arabidopsis thaliana seedlings as a model system to evaluate the effects of melatonin on plant growth and development. Melatonin modulated root system architecture by stimulating lateral and adventitious root formation but minimally affected primary root growth or root hair development. The auxin activity of melatonin in roots was investigated using the auxin-responsive marker constructs DR5:uidA, BA3:uidA, and HS::AXR3NT-GUS. Our results show that melatonin neither activates auxin-inducible gene expression nor induces the degradation of HS::AXR3NT-GUS, indicating that root developmental changes elicited by melatonin were independent of auxin signaling. Taken together, our results suggest that melatonin is beneficial to plants by increasing root branching and that root development processes elicited by this novel plant signal are likely independent of auxin responses.