Mango (2n = 2x = 40) is an important tropical/subtropical evergreen fruit tree grown worldwide, and its fruits are nutritionally rich and of high value. Here, a high-quality mango genome (396 Mb, contig N50 = 1.03 Mb) was assembled using the cultivar ‘Irwin’ from Florida, USA. A total of 97.19% sequences were anchored to 20 chromosomes, including 36,756 protein-coding genes. We compared the β-carotene content, in two different cultivars and growth periods, and the variation of β-carotene content mainly affected the fruit flesh colour. Also, from their transcriptome analysis, the β-carotene biosynthesis genes were identified. MiPSY1was proved to be an key gene regulating the β-carotene biosynthesis. Weighted gene co-expression network analysis (WGCNA), dual luciferase and yeast one-hybrid assays confirmed that transcription factors (TFs) MibZIP66 and MibHLH45 activate MiPSY1 transcription by directly binding to the CACGTG motif of MiPSY1 promoter. However, the two TFs showed no significant synergistic effects on promoter activity. Taken together, the current study provides a genomic platform for studying the molecular basis of fruit flesh color in mango.