Host-selective ACT toxin are critical for the pathogenesis of the citrus fungal pathogen Alternaria alternata. The biosynthesis of ACT toxin is mainly regulated by multiple ACT toxin genes located in the secondary metabolite gene cluster. However, the regulatory hierarchy of ACT toxin synthesis by these ACT genes have not been explored. In this study, we reported a transcription regulator ACTR contributes ACT toxin biosynthesis through mediating ACT toxin synthesis gene ACTS4 in Alternaria alternata. We generated ACTR-disrupted and -silenced mutants in the tangerine pathotype of A. alternata. Phenotype analysis showed that the ACTR mutants displayed a significant loss of ACT toxin production and a decreased virulence on citrus leaves whereas the vegetative growth and sporulation were not affected, indicating an essential role of ACTR in both ACT toxin biosynthesis and pathogenicity. To elucidate the transcription network of ACTR, we performed RNA-Seq experiments on wild-type and ACTR null mutant and identified genes that were differentially expressed between two genotypes. Transcriptome profiling and RT-qPCR analysis demonstrated that the ACT toxin biosynthetic gene ACTS4 is down-regulated in ACTR mutant. We generated ACTS4 knock-down mutant and found that the pathogenicity of ACTS4 mutant was severely impaired. Interestingly, both ACTR and ACTS4 are not involved in the response to different abiotic stresses including oxidative stress, salt stress, cell-wall disrupting regents, and Cu2+, indicating the function of these two genes is highly specific. In conclusion, our results highlight the important regulatory role of ACTR in ACT toxin biosynthesis through mediating ACT toxin synthesis gene ACTS4 and underline the essential role of in the tangerine pathotype of A. alternata.