GABAB, μ‐opioid and adrenergic α2 receptors inhibit substance P release from primary afferent terminals in the dorsal horn. Studies in cell expression systems suggest that μ‐opioid and GABAB receptors inhibit transmitter release from primary afferents by activating Src family kinases (SFKs), which then phosphorylate and inhibit voltage‐gated calcium channels. This study investigated whether SFKs mediate the inhibition of substance P release by these three receptors. Substance P release was measured as neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo. In slices, NK1R internalization induced by high‐frequency dorsal root stimulation was inhibited by the μ‐opioid agonist DAMGO and the GABAB agonist baclofen. This inhibition was reversed by the SFK inhibitor PP1. NK1R internalization induced by low‐frequency stimulation was also inhibited by DAMGO, but PP1 did not reverse this effect. In vivo, NK1R internalization induced by noxious mechanical stimulation of the hind paw was inhibited by intrathecal DAMGO and baclofen. This inhibition was reversed by intrathecal PP1, but not by the inactive PP1 analog PP3. PP1 produced no effect by itself. The α2 adrenergic agonists medetomidine and guanfacine produced a small but statistically significant inhibition of NK1R internalization induced by low‐frequency dorsal root stimulation. PP1 did not reverse the inhibition by guanfacine. These results show that SFKs mediate the inhibition of substance P release by μ‐opioid and GABAB receptors, but not by α2 receptors, which is probably mediated by the binding of G protein βγ subunits to calcium channels.