Abstract: This study aims to investigate the potential mechanism of melatonin (MT) alleviating the browning of postharvest lotus seeds. Firstly, in order to explore the possible pathway of NO biosynthesis in the lotus seeds, the samples were treated with 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. Then, the changes in the levels of key enzymes and substances involved in the NO biosynthesis was analyzed. The results showed that the endogenous NO content of the MT treated lotus seeds was significantly higher than that of the control, and 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 are the key substances involved in the NO biosynthesis, when compared with the control. 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, the influence of MT treatment on the activity of NR in lotus seeds was not consistent, and the beneficial effect of MT on the lotus seeds and the endogenous NO content of the tissue were not affected 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, and the endogenous NO content in this treatment was significantly lower than that in the control. Therefore, it was deduced that the exogenous MT treatment induced the NO biosynthesis in lotus seeds through the pathway of NOS. Secondly, to investigate the potential mechanism of MT regulating the process of browning of lotus seeds, the phenolic compounds in the lotus seeds were identified 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. These results suggested that the catechin was the major phenolic compound of lotus seeds. Literatures have confirmed that the flavan-3-ol was the most common direct natural substrate of polyphenol oxidase (PPO) in the plant, and especially for the catechin and epicatechin. Thereby, this research focused on the potential mechanism of MT regulating the catechin metabolism through inducing 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 groups. 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), which was involved 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 groups 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 induced the biosynthesis of NO through NOS pathway, and then the accumulated NO acted on the catechin metabolism of lotus seeds. On one hand, the MT treatment suppressed the catechin to participate in the enzymatic browning by inhibiting the PPO activity; on the other hand, the biosynthesis of catechin was promoted by this treatment. Consequently, the process of the lotus seeds browning was alleviated by the MT treatment. These findings can provide the theoretical and technical support for preservation of lotus seeds and the signal transduction between MT and NO.