There is a higher incidence of local anesthetic failure in endodontic patients experiencing pulpal hyperalgesia. Up-regulation of Nav1.9, a voltage-gated sodium channel isoform, might play a key role in local anesthetic failure because Nav1.9 channels increase neuronal excitability and have low sensitivity to blockade by local anesthetics. Immunocytochemistry was used to examine Nav1.9 channel expression in axons of symptomatic (painful) versus asymptomatic human dental pulp and to determine Nav1.9 expression levels in neuronal somata of the human trigeminal ganglion. Nav1.9 channel immunoreactivity on pulpal axons was significantly increased in painful teeth. Nav1.9 channels were expressed in membranes and cytoplasm of human trigeminal ganglion neurons, with the highest expression in small neuronal somata. Nav1.9 expression in the trigeminal ganglion coupled with increased expression in symptomatic pulp might contribute to hypersensitivity of inflamed pulps and local anesthetic failure. Furthermore, the present study suggests that Nav1.9 channels are potential targets for novel anesthetics.