Hypocrellins, as natural pigments from Shiraia bambusicola, have extensive applications in the agricultural, cosmetic, food, and feed industries, and play a vital role in photodynamic therapy for anticancer and antiviral treatments. However, environmental stresses are always the bottlenecks for increasing hypocrellin yield during the process of fermentation. Pre-mRNA alternative splicing (AS) is an essential mechanism in the defense of abiotic stresses in the animal and plant kingdom, but is seldom involved in fungi. In this study, AS from genome-wide sequencing and RNA-seq data for S. bambusicola was analyzed for the first time. Interestingly, the proportion of AS in S. bambusicola was 38.44% (most of them participated in metabolic processes, covering pigmentation and response to stimulus), a much higher ratio than seen in that of other fungal species (1.3-18%). Here, we identified the relationship of AS and secondary metabolic (SM) biosynthesis under a series of abiotic stresses. Suitable fungicides suppressed hypocrellin production significantly, and AS occurred in key functional genes (sbFLO, sbMFS, sbPKS) of hypocrellin biosynthesis. In contrast, H2O2 improved the yield of hypocrellins, but AS were not found in the corresponding gene cluster. A further study showed that overexpressing an isoform of sbPKS (sbPKSa) in Shiraia bambusicola could dramatically down-regulate the expression of the original gene sbPKS and nearly inhibit the production of hypocrellins. Altogether, our study strongly supported the hypothesis that AS had a vital role in the regulation of hypocrellin biosynthesis under stresses, and initially explored whether SM functional genes were relevant for fungi.