Rab GTPases are the largest group of the small GTPases family, which play a pivotal role in the secretion of proteins. Arthrobotrys oligospora is a representative nematode-trapping fungus that can produce adhesive networks to capture nematodes. In this study, the roles of two Rab GTPases AoRab-7A and AoRab-2 were characterized by gene knockout in the fungus A. oligospora. The disruption of AoRab-7A hindered the mycelial growth in different media, the conidiation of Delta AoRab-7A transformants was almost abolished, and the transcription of four sporulation-related genes (AbaA, FluG, Hyp1, and VosA) was downregulated compared to the wild-type strain (WT). Furthermore, the tolerance of the Delta AoRab-7A mutants to sodium dodecyl sulfate (SDS) and H2O2 was also significantly reduced compared to the WT, and the transcription of several genes related to environmental resistance, such as genes for catalase and trehalose synthase, was downregulated. Similarly, the extracellular proteolytic activity was decreased. Importantly, the Delta AoRab-7A mutants were unable to produce traps and capture nematodes. However, the disruption of gene AoRab-2 only affected the conidiation slightly but non-significantly, while other phenotypic traits were unaffected. Moreover, the gene AoRab-7A was also involved in the autophagy induced by nitrogen deprivation in A. oligospora. Our results revealed for the first time that the Rab GTPases are involved in the regulation of mycelial growth, conidiation, trap formation, stress resistance, and pathogenicity in the nematode-trapping fungus A. oligospora.