AbstractHypocrellin A (HA), a promising photosensitizer for anticancer photodynamic therapy (PDT), is a fungal perylenequinone pigment from the fruiting body of Shiraia bambusicola, a traditional Chinese medicine for treating skin diseases. The mycelial cultures are becoming a biotechnological alternative for HA production. In this study, light of different wavelengths was investigated to develop an effective eliciting strategy for HA production in the cultures. Under red LED light (627 nm) at 200 lux, the maximum HA production (175.53 mg L−1) in mycelium cultures was reached after 8 days, about 3.82‐fold of the dark control. Red light not only promoted HA biosynthesis in mycelia (intracellular HA), but also stimulated HA secretion into the medium (extracellular HA). We found 14 of 310 transcripts differentially expressed under red light treatment were possible candidate genes for HA biosynthetic pathway. Gene ontology (GO) analysis revealed that red light treatment could change the gene expressions responsible for HA biosynthesis and the transmembrane activity, suggesting both intracellular HA and its secretion could contribute to the enhancement of total HA production in the cultures. The results provided new insights of red light elicitation and effective strategy for HA production in mycelium cultures.