The study was aimed to explore the molecular mechanism of detoxification of prochloraz by Lasiodiplodia theobromae from mango and to screen related functional genes. In this study, RNA-seq technology was used to analyze the transcriptome sequencing and functional annotation of prochloraz-treated and untreated strains of L. theobromae. Prochloraz had significant inhibitory activity against the sensitive strain with an EC₅₀ value of 0.12 mg/L. The EC₅₀ value of the two resistant strains was 7.67 and 32.67 times higher than that of the sensitive strain, respectively. GO functional enrichment and KEGG signaling pathway enrichment analyses were performed for differentially expressed genes (DEGs) between prochloraz-sensitive strains (DF04) and -resistant strains (HL02 and M108). Compared with the untreated (CK) with prochloraz, there were 5342, 1148, and 2568 DEGs in DF04, HL02, and M108, respectively, after 10 mg/L prochloraz treatment. Among them, a total of 566 DEGs were overlapped among the three strains. Compared with the sensitive strains, the resistant strains had 2078 and 3096 DEGs up-regulated and 1527 and 1793 down-regulated expression, respectively. GO enrichment showed that the highest number of genes were involved in cellular process and metabolic process between resistant and sensitive strains after prochloraz treatment, accounting for 18% and 16%, 17% and 15% in HL02 and M108, respectively. KEGG enrichment showed that DEGs were significantly enriched in metabolic pathways between resistant and sensitive strains after prochloraz treatment, with 64.98% and 63.39% in HL02 and M108, respectively. The analysis for DEGs related to detoxification metabolism identified 27, 17 and 59 cytochrome P450 (Cytochrome P450), glutathione transferase (GST) and ABC transporter protein (ABC transporter) genes, respectively. Eight genes were randomly screened for quantitative real-time PCR validation, and the results confirmed that the trends in the relative expression of these genes were consistent with the transcriptome data. Based on the significantly up-regulated expression of DEGS in the resistant strains, the final screening of 7 P450s genes, 8 GSTs genes and 11 ABC transporter protein genes may be related to the metabolic resistance mechanism of L. theobromae to prochloraz. The results would provide new insights into the mechanism of fungicide resistance and lay the foundation for in-depth analysis of the molecular mechanism of metabolic resistance to prochloraz.