Cannabidiol (CBD), the main nonpsychoactive cannabinoid in , has diverse applications in the pharmacological, food, and cosmetic industries. The long plantation period and the complex chemical structure of cannabidiol pose a great challenge on CBD supply. Here, we achieved biosynthesis of cannabidiol in . The CBD production was further enhanced by 2.53-fold through pushing the supply of precursors and fusion protein construction. Bile pigment transporter 1 (BPT1) was the most effective transporter for transferring cannabigerolic acid (CBGA) from the cytoplasm to the vacuole, which removed the physical barrier separating CBGA and its catalytic enzyme. The lowest binding energy of the CBGA-BPT1 complex confirmed a strong interaction between BPT1 and CBGA. A CBD yield of 6.92 mg/L was achieved, which was 100-fold higher than the yield generated by the starting strain. This study provides insights into high-level CBD-producing strain construction and lays the foundation for CBD supply.