The agouti signaling protein gene (ASIP) is a widely studied pigmentation gene that plays an important role in melanin synthesis. To determine the variety of ASIP expression in the Muchuan Black‐Bone chicken, we examined genetic variation in the ASIP promoter region. A single nucleotide polymorphism (c.–1826A>T) was found to be associated with the skin color (dorsal and subalar) of black‐bone chicken. Individuals with TT and AT genotypes had higher ASIP mRNA levels in the skin than did those with the AA genotype (P < 0.01). In addition, individuals with the TT genotype had higher ASIP mRNA levels than did those with the AT genotype (P < 0.05). Expression of melanogenesis‐related genes (melanocortin 1 receptor and tyrosinase genes) was higher in the skin of chickens with the TT and AT genotypes than in those with the AA genotype (P < 0.01). A luciferase assay showed that promoter activity was higher in chickens with the TT genotype than in those with the AA genotype. Putative transcription factor prediction suggested that the c.–1826A>T mutation might shift the promoter binding affinity with differential transcription factors. In summary, we identified a novel mutation in the ASIP gene promoter that may affect chicken skin color by altering ASIP transcriptional activity.