The anthracnose rot of postharvest mango fruit is a devastating fungal disease often resulting in tremendous quality deterioration and postharvest losses. Nitric oxide (NO), as an important signaling molecule, is involved in the responses to postharvest fruit diseases. In the present study, the effectiveness of NO donor sodium nitroprusside (SNP) to prevent anthracnose of "Tainong" mango fruit caused by Colletotrichum gloeosporioides was evaluated through in vivo and in vitro tests. Results from in vivo test showed that SNP treatment effectively inhibited the lesion diameter and disease incidence on inoculated mango fruit during storage. SNP treatment could regulate hydrogen peroxide levels by reinforcing the activities of catalase, peroxidase, superoxide dismutase, and ascorbate peroxidase. Furthermore, SNP elevated the accumulation of lignin, total phenolics, anthocyanin, and flavonoids and the activities of chitinase and β-1,3-glucanase. In addition, in vitro tests indicated that SNP markedly suppressed mycelial growth and spore germination of C. gloeosporioides through damaging plasma membrane integrity and increasing the leakage of soluble sugar and protein. Our results suggested that SNP could suppress anthracnose decay in postharvest mango fruit, possibly by directly suppressing pathogen growth and indirectly triggering host defense responses.