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Excessive aluminum ions (Al3+) in acidic soil can have a toxic effect on watermelons, restricting plant growth and reducing yield and quality. In this study, we found that exogenous application of nitric oxide (NO) could increase the photochemical efficiency of watermelon leaves under aluminum stress by promoting closure of leaf stomata, reducing malondialdehyde and superoxide anion in leaves, and increasing POD and CAT activity. These findings showed that the exogenous application of NO improved the ability of watermelon to withstand aluminum stress. To further reveal the mitigation mechanism of NO on watermelons under aluminum stress, the differences following different types of treatments—normal growth, Al, and Al + NO—were shown using de novo sequencing of transcriptomes. In total, 511 differentially expressed genes (DEGs) were identified between the Al + NO and Al treatment groups. Significantly enriched biological processes included nitrogen metabolism, phenylpropane metabolism, and photosynthesis. We selected 23 genes related to antioxidant enzymes and phenylpropane metabolism for qRT-PCR validation. The results showed that after exogenous application of NO, the expression of genes encoding POD and CAT increased, consistent with the results of the physiological indicators. The expression patterns of genes involved in phenylpropanoid metabolism were consistent with the transcriptome expression abundance. These results indicate that aluminum stress was involved in the inhibition of the photosynthetic pathway, and NO could activate the antioxidant enzyme defense system and phenylpropane metabolism to protect cells and scavenge reactive oxygen species. This study improves our current understanding by comprehensively analyzing the molecular mechanisms underlying NO-induced aluminum stress alleviation in watermelons. [ABSTRACT FROM AUTHOR]