Summary Aspergillus fumigatus is a fungal pathogen that is capable of adapting to different host niches and to avoid host defenses. An enhanced understanding of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes is essential for the development of improved disease control strategies. Protein phosphatases are central to numerous signal transduction pathways. To comprehend the functions of protein phosphatases in A. fumigatus, 32 phosphatase catalytic subunit encoding genes were identified. We have recognized PtcB as one of the phosphatases involved in the high osmolarity glycerol response (HOG) pathway. The ΔptcB mutant has both increased phosphorylation of the p38 MAPK (SakA) and expression of osmo‐dependent genes. The ΔptcB strain was more sensitive to cell wall damaging agents, had increased chitin and β‐1,3‐glucan, and impaired biofilm formation. The ΔptcB strain was avirulent in a murine model of invasive pulmonary aspergillosis. These results stress the importance of the HOG pathway in the regulation of pathogenicity determinants and virulence in A. fumigatus. Aspergillus fumigatus is a fungal pathogen that is capable of adapting to different host niches and avoiding host defenses. We have recognized PtcB as a protein phosphatase involved in the HOG (High Osmolarity Glycerol response) pathway. The ΔptcB mutant has both increased phosphorylation of the p38 MAPK (SakA) and expression of osmo‐dependent genes. The ΔptcB strain was avirulent in a murine model of invasive pulmonary aspergillosis, stressing the importance of the HOG pathway in the regulation of pathogenicity and virulence.