In addition to its well-known effects on parturition and lactation, oxytocin (OT) plays an important role in modulation of pain and nociceptive transmission. But, the mechanism of this effect is unclear. To address the possible role of OT on pain modulation at the peripheral level, the effects of OT on intracellular calcium levels ([Ca(2+)](i)) in rat dorsal root ganglion (DRG) neurons were investigated by using an in vitro calcium imaging system. DRG neurons were grown in primary culture following enzymatic and mechanical dissociation of ganglia from 1- or 2-day-old neonatal Wistar rats. Using the fura-2-based calcium imaging technique, the effects of OT on [Ca(2+)](i) and role of the protein kinase C (PKC)-mediated pathway in OT effect were assessed. OT caused a significant increase in basal levels of [Ca(2+)](i) after application at the doses of 30 nM (n = 34, p < 0.01), 100 nM (n = 41, p < 0.001) and 300 nM (n = 46, p < 0.001). The stimulatory effect of OT (300 nM) on [Ca(2+)](i) was persistent in Ca(2+)-free conditions (n = 56, p < 0.01). Chelerythrine chloride, a PKC inhibitor, significantly reduced the OT-induced increase in [Ca(2+)](i) (n = 28, p < 0.001). We demonstrated that OT activates intracellular calcium signaling in cultured rat primary sensory neurons in a dose- and PKC-dependent mechanism. The finding of the role of OT in peripheral pain modification may serve as a novel target for the development of new pharmacological strategies for the management of pain.