ABSTRACT: Melatonin has been regarded as a promising substance that enhances the abiotic stress tolerance of plants. However, few studies have devoted attention to the role of melatonin in improving salt tolerance in sugar beets. Here, the effects of different application methods (foliar application (100 μM), root application (100 μM), and combined foliar and root application) of melatonin on the morphological and physiological traits of sugar beets exposed to salt stress were investigated. The results showed that melatonin improved the growth of sugar beet seedlings, root yield and sugar content, synthesis of chlorophyll, photosystem II (PS II) activity, and gas exchange parameters under salt stress conditions. Moreover, melatonin enhanced the capacity of osmotic adjustment by increasing the accumulation of osmolytes (betaine, proline, and soluble sugar). At the same time, melatonin increased the H+-pump activities in the roots, thus promoting Na+ efflux and K+ influx, which maintained K+/Na+ homeostasis and mitigated Na+ toxicity. In addition, melatonin strengthened the antioxidant defense system by enhancing the activities of antioxidant enzymes, modulating the ASA-GSH cycle, and mediating the phenylalanine pathway, which removed superoxide anions (O2•-) and hydrogen peroxide (H2O2) and maintained cell membrane integrity. These positive effects were more pronounced when melatonin was applied by combined foliar and root application. To summarize, this study clarifies the potential roles of melatonin in mitigating salt stress in sugar beets by improving photosynthesis, water status, ion homeostasis, and the antioxidant defense system.