Hog1, a mitogen-activated protein kinase (MAPK), has been identified in diverse fungal species, and it regulates various cellular processes, such as osmoadaptation, nutrient-sensing, and pathogenesis. However, the roles that Hog1 plays in nematode-trapping fungi were previously unclear. Here, we characterized orthologs of Saccharomyces cerevisiae Hog1 and membrane mucin Msb2 in the nematode-trapping fungus Arthrobotrys oligospora. We generated gene deletion mutants of HOG1 and MSB2 in A. oligospora, and characterized their roles in osmosensing, growth, and trap morphogenesis. We found that both hog1 and msb2 mutants were highly sensitive to high osmolarity. Predation analyses further revealed that hog1 and msb2 deletion caused a reduction in trap formation and predation efficiency. Furthermore, HOG1 is required for conidiation in A. oligospora, demonstrating its critical role in this developmental pathway. In summary, this study demonstrated that the conserved Hog1 and Msb2 govern physiology, growth and development in the nematode-trapping fungus A. oligospora.