Root growth and development is an important indicator of root-zone hypoxia tolerance in rice. Melatonin has been suggested to function as a crucial regulator in modulating root growth and improving plant abiotic stress resistance. To explore the role and potential mechanism of melatonin in regulating the root growth under root-zone hypoxia stress, rice seedlings were treated with hypoxia (oxygen level at 0.9–2.1 mg·L−1), combined with or without a 20 μmol·L−1 melatonin pretreatment under a hydroponic condition. The results showed that the exogenous application of melatonin significantly alleviated the inhibition of the rice root growth that was induced by the hypoxia stress. The morphological–phenotypic analyses showed that after the melatonin pretreatment, the primary root length, lateral root length, and lateral root density increased by 11.6%, 8.2%, and 36.8%, respectively, under hypoxia stress. The physiological–biochemical analyses showed that the exogenous melatonin significantly increased the root activity and O2 influx in the root meristem zone under hypoxia stress to 1.5 times that observed in the hypoxia stress group. The melatonin pretreatment significantly improved the activity of superoxide dismutase (SOD) and decreased the accumulation of superoxide anions (O2•−) in the seedling roots, whereas it increased the content of hydrogen peroxide (H2O2) under hypoxia stress. The exogenous melatonin pretreatment significantly increased the content of indole-3-acetic acid (IAA) by 51.5% in the rice roots compared to the plants without melatonin pretreatment under hypoxia stress. Quantitative real-time PCR (qRT-PCR) analyses revealed that the melatonin pretreatment induced the expression of OsPIN1a~1d, OsPIN8, OsPIN9, OsAUX1, OsARF19, and OsGH3-2 in the rice seedling roots under aerated conditions, whereas it only obviously upregulated the expression of OsPIN1b, OsPIN2, and OsGH3-2 under hypoxia stress. These results indicate that melatonin positively regulates root growth and development under hypoxia stress, through improving the antioxidant system and directly or indirectly activating the auxin signaling pathway. This study demonstrates the important role of melatonin to modulate root growth under hypoxia stress, providing a new strategy for improving hypoxia tolerance.