Melatonin is widely involved in plant disease resistance through modulation of immune responses. Pathogenesis-related (PR) proteins play important roles in plant immune responses. However, the direct association between melatonin biosynthetic enzyme and PR protein remains elusive in plants. In this study, we found that N-aceylserotonin O-methyltransferase 2 (MeASMT2) physically interacted with MePR1 in vitro and in vivo, thereby promoting the anti-bacterial activity of MePR1 against Xanthomonas axonopodis pv. manihotis (Xam). Consistently, MeASMT2 improved the effect of MePR1 on positively regulating cassava disease resistance. In addition, we found that type 2C protein phosphatase 1 (MePP2C1) interacted with MeASMT2 to interfere with MePR1-MeASMT2 interaction, so as to inhibiting the effect of MeASMT2 and MePR1 on positively regulating cassava disease resistance. In contrast to the increased transcripts of MeASMT2 and MePR1 in response to Xam infection, the transcript of MePP2C1 was decreased upon Xam infection. Therefore, disease activated MeASMT2 was released from disease inhibited MePP2C1, so as to improving the anti-bacterial activity of MePR1, resulting in improved immune response. In summary, this study illustrates the dynamic modulation of the MePP2C1-MeASMT2-MePR1 module on cassava defense response against cassava bacterial blight (CBB), extending the understanding of the correlation between melatonin biosynthetic enzyme and PR in plants.