Updated project metadata. Cryptococcus neoformans is a life-threatening basidiomycete fungal pathogen responsible for meningoencephalitis in immunocompromised patients. This yeast can adapt to diverse habitats, efficiently produces virulence factors, and escapes immune surveillance. This implies intricate mechanisms underlying its gene regulation networks, which are yet to be comprehensively understood. Alternative transcription usage regulation has been identified as the major mean for gene expression regulation in metazoans. However, in fungi, its impact remains elusive as its study has thus far been restricted to model yeasts. We here re-analysed transcription start site (TSS)-seq data to define genuine TSS clusters in two species of pathogenic Cryptococcus. We identified two types of TSS clusters associated with specific DNA sequence motifs. Our analysis also revealed that alternative TSS usage regulation in response to environmental cues is widespread in Cryptococcus, altering gene expression and protein targeting. Importantly, we performed a forward genetic screen to identify a unique transcription factor (TF) named Tur1, which regulates aTSS usage genome-wide when cells switch from exponential phase to stationary phase. Tur1 has been previously shown to be essential for virulence in C. neoformans. Accordingly, we demonstrated here that a tur1Δ mutant strain is more sensitive to superoxide stress and phagocytosed more efficiently by macrophages than the Wild-type (WT) strain.