The photoinduced β-carotene synthesis in Blakeslea trispora is dependent on WC-2A

β-Carotene, a high value-added natural pigment, is currently produced industrially in <i>Blakeslea trispora</i>. Although photoinduced carotenoid synthesis has been identified in some filamentous fungi, there are still relatively few studies focusing on <i>B. trispora</i> and its potential mechanisms. In this study, an integrated strategy-including correlation analysis of gene expression, bioinformatics analysis, protein interaction, and RNA interference-was adopted to elucidate photoinduced β-carotene synthesis in <i>B. trispora</i>. Light wavelength, intensity, and irradiation duration stimulated the transcription of photoreceptors [<i>btwc-1</i> (<i>a, b, c</i>) and <i>btwc-2</i> (<i>a, b, c, d</i>)] and carotenoid structural genes (<i>carB</i> and <i>carRA</i>). The transcription of photoreceptor genes showed significant or high correlation with carotenoid structural genes under continuous or short-term, high-intensity blue light irradiation. To elucidate the role of photoreceptors in carotenoid synthesis, the interaction between BTWC-1 and BTWC-2 was predicted. Furthermore, Glutathione S-Transferase (GST) pull-down assays showed that only BTWC-1C and BTWC-2A could interact to form complexes. Inhibition of <i>btwc-2a</i> expression under dark conditions did not affect β-carotene accumulation or the transcription of <i>carB</i> and <i>carRA</i>, but did reduce these parameters under blue light irradiation, indicating that <i>btwc-2a</i> mediates photoinduced β-carotene synthesis in <i>B. trispora</i>.