This study aims to investigate the potential mechanism of melatonin (MT) to alleviate the browning of postharvest lotus seeds. 1) The samples were treated with the distilled water, nitric oxide (NO), MT, the inhibitor of nitric oxide synthase (NOS) nomega-nitro-L-arginine methyl ester (L-NAME), the inhibitor of nitrate reductase (NR) tungstate (TUN), and the scavenger of NO carboxy-PTIO (cPTIO), MT + L-NAME, MT + TUN and MT + cPTIO. A systematic analysis was implemented to explore the possible pathway of NO biosynthesis in the lotus seeds. Then, the levels of key enzymes and substances were determined to involve in the NO biosynthesis. The results showed that the endogenous NO content of the MT treated lotus seeds was significantly higher than that of the control. The NOS activity of lotus seeds treated with MT was promoted by 60.36%~71.08% before 3 days of storage. In addition, the MT treatment significantly increased the contents of L-arginine and citrulline (P <0.05), which were the key substances in the NO biosynthesis. It was notable that the citrulline content in the MT treated sample was 1.44~1.59 times than those of the controls. By contrast, there was the varying influence of MT treatment on the activity of NR in lotus seeds. There was the beneficial effect of MT on the lotus seeds and the endogenous NO content of the tissue, when the MT was combined with the inhibitor of NR (TUN). However, this beneficial effect disappeared, when the MT was combined with the inhibitor of NOS (L-NAME) for treating the lotus seeds. The endogenous NO content in this treatment was significantly lower than that in the control. Therefore, the exogenous MT treatment was induced the NO biosynthesis in lotus seeds through the pathway of NOS. 2) The potential mechanism of MT was clarified to regulate the browning of lotus seeds. The phenolic compounds were identified in the lotus seeds using a LC20 HPLC (Shimadzu, Japan) system coupled to a TripleTOF? 5600 + quadrupole time-of-flight (QTOF) mass spectrometer equipped with a DuoSpray? ion source (Sciex, Ontario, Canada). It was found that there were six flavanols and seven phenolic acids in the lotus seeds, and the content of catechin, a type of flavanol, accounts for over 60% of the total phenol content, the following was ellagitannin, a type of phenolic acid, accounts for 7.45% of the total phenol content. It infers that the catechin was the major phenolic compound of lotus seeds. The flavan-3-ol was the most common direct natural substrate of polyphenol oxidase (PPO) in the plant, especially for the catechin and epicatechin. Thereby, the potential mechanism of MT was determined, where the catechin metabolism was regulated to induce the NO production in the following experiments. The results indicated that the activity of PPO in the MT treated lotus seeds was lower by 35.39%~57.36% than those in the control. What’s more, the MT treatment significantly increased the activities of key enzymes, including cinnamate-4-hydroxylase, dihydroflavonol reductase, chalcone synthase, chalcone isomerase and colorless anthocyanin reductase (P <0.05) in the catechin synthetic metabolism. As a result, the content of catechin in the MT treated sample was higher by 15.35%~47.86% than those in the control after 1 days of storage. Whereas, this positive effect was negated, when the MT was combined with L-NAME or cPTIO for treating lotus seeds. Therefore, it was concluded that the exogenous MT treatment was induced the biosynthesis of NO through NOS pathway, and then the accumulated NO acted on the catechin metabolism of lotus seeds. The MT treatment was used to inhibit the PPO activity, and then the catechin was suppressed to participate in the enzymatic browning; The biosynthesis of catechin was promoted in this treatment. Consequently, the lotus seeds browning was also alleviated by the MT treatment. These findings can provide the theoretical and technical support to preservation of lotus seeds and the signal transduction between MT and NO.