In these studies, we collected American dog ticks (Dermacentor variabilis) from four natural populations within Manitoba, Canada. We first carried out behavior assays, which showed DEET effectively repelled the ticks. RNA sequencing revealed that DEET caused a rapid and substantial reduction in the abundance of transcripts encoding cytochrome P450 and acetylcholinesterase genes, which gradually recovered over the 24 h time course. Finally, enzymatic kinetics provided functional support for DEET's role as an effective inhibitor of P450 s. While many facets of its mode of action remain to be worked out, our study provides valuable insights into the molecular underpinnings of DEET's repellence in ticks.