The bacterial pathogen Pseudomonas syringae depends on effector proteins secreted by its type III secretion system for the pathogenesis of plants. The majority of these effector proteins are known suppressors of immunity, but their plant targets remain elusive. Using Arabidopsis thaliana as a model host, we report that the HopE1 effector uses the host calcium sensor, calmodulin (CaM), as a co-factor to target the microtubule-associated protein 65 (MAP65), an important component of the microtubule network. HopE1 interacted with MAP65 in a CaM-dependent manner, resulting in MAP65-GFP dissociation from microtubules. Transgenic Arabidopsis expressing HopE1 had reduced secretion of the immunity protein PR-1 compared to wild-type plants. Additionally, Arabidopsis map65-1 mutants were immune deficient and were more susceptible to P. syringae. Our results suggest a virulence strategy in which a pathogen effector is activated by host calmodulin to target MAP65 and the microtubule network, thereby inhibiting cell wall-based extracellular immunity.