Background and Purpose: The intranasal administration of oxytocin (OT) reduces migraine headaches through activation of the oxytocin receptor (OTR). Magnesium ion (Mg2+) concentration is critical to activation of OTR, and low serum Mg2+ concentration is predictive of migraine headache. We, therefore, examined the functional impact of Mg2+ concentration on OT-OTR binding efficacy using two complimentary bioassays. Current clamp recordings of rat trigeminal ganglia neurons (TG) measured the impact of Mg2+ on OT-induced reduction in excitability. Secondly, we assessed the impact of Mg2+ on intranasal OT-induced craniofacial analgesia in rats. Key Results: OT alone dose-dependently hyperpolarized TG neurons, decreasing their excitability; the addition of 1.75mM Mg2+ significantly enhanced this effect. Similarly, while intranasal application of OT produced dose-dependent craniofacial analgesia; Mg2+ significantly enhanced these effects. Conclusion and Implications: Mg2+ concentration is critical to OT-OTR signaling, and OT efficacy may be limited by low ambient Mg2+ levels. The addition of Mg2+ to OT formulations may improve its efficacy in reducing headache pain as well as for other oxytocin-dependent processes.