Opiorphin is an endogenous human peptide mediator constituent of human saliva. Initial research with mice shows opiorphin has highly effective analgesic activity than morphine. Effect of opiorphin on capsaicin sensitive sensory neurons was investigated by using in-vitro calcium imaging technique in this study. Dorsal root ganglions (DRGs) were removed from 1-2 day old (any gender) Wistar neonatal rats after decapitation and DRG neurons were grown in primary culture following enzymatic and mechanical dissociation and cultured in a tissue culture containing nerve growth factor for a short time. Neurons were loaded with 1μmol Fura-2 AM. After loading, the cells were washed 3-4 times for 20min with standard recording medium to remove the extracellular fura-2 AM. [Ca2+]i responses were quantified by the changes in 340/380 ratio by using fluorescence imaging system. Based on the size of cell trunk, DRG neurons were divided into three categories: small, medium and large. Responses to capsaicin (1μM) (small and medium size) was studied by monitoring changes in [Ca2+]i with a microscobic digital image analysis system in fura-2 loaded single neurons. We tested the effects of different concentration of opiorphin treatment after recording of basal [Ca2+]i. The application of 10μM,100μM and 1000μM opiorphin had significant decrease on capsaicin evoked [Ca2+]i to 99.4±2.0% (n = 48), 51.2±5.8% (n=38, P<0.001) and 2.5±1.2% (n=24, respectively, P<0.0001) in small size neurons. Same doses of opiorphin had significant decrease on capsaicin evoked [Ca2+]i to 99.1±2.3% (n=36), 53.4±5.8% (n=15, P<0.001) and 7.6±1.9% (n=15, respectively, P<0.0001) in medium size cells. Data from this study revealed that opiorphin inhibits intracellular calcium signaling in cultured rat DRG neurons in a dose dependent manner. Further studies are needed to find out the efficency of opiorphin on pain modulation.