Oxytocin (OT), composed of nine amino acids, has a wide range of physiological functions such as uterine contractions, maternal/social behavior and anti-stress effects. Recently, it has been reported that OT released from parvocellular neurons alleviates pain. Analgesic effects of OT might be involved in the μ- and κ-opioid receptors (OR) in the rats because it was partially inhibited by the antagonists of μ- and κ-ORs but not δ-ORs. However, it has not been elucidated the mechanism in detail. While we previously reported that OT could enhance the μ-OR activity as a positive allosteric modulator (PAM), it remains unclear whether OT behaves as a PAM for the δ- and κ-OR. We therefore analyzed activities of δ- and κ-OR stimulated by OT in two types of HEK293 cells stably expressing δ- and κ-OR, respectively. For the measurement, we used the CellKey^TM assay system that measures changes of impedance following OR activation. In the assay, OT failed to exert agonistic effects on the δ- and κ-OR, whereas it enhanced the κ-OR activity induced by κ-OR agonists dynorphin A, U50488 and morphine. Interestingly, OT at 10^-6 M had no effect on the δ-OR activity induced by δ-OR agonists Leu- and Met-enkephalin, SNC80 and morphine. We also revealed all amino acids of OT except Leu in the position 8 could be involved in PAM activity at the κ-OR. In addition, our competitive receptor-binding analysis disclosed that OT had no effect on the κ-OR orthosteric binding sites. Hence, OT could function as a κ-OR PAM in addition to a μ-OR PAM.