We assessed the effects of tramadol, a centrally acting analgesic, and its major metabolite, on neurotransmitter-gated ion channels. Tramadol binds to μ-opioid receptors with low affinity and inhibits reuptake of monoamines in the central nervous system. These actions are believed to primarily contribute to its antinociceptive effects. However, little is known about other sites of tramadol's action. We tested the effects of tramadol and its M1 metabolite (0.1–100 μM) on human recombinant neurotransmitter-gated ion channels, including glycine, γ-aminobutyric acidA (GABAA), and N-methyl-d-aspartate (NMDA) receptors, expressed in Xenopus oocytes. Tramadol and M1 metabolite did not have any effects on glycine receptors. GABAA receptors were significantly inhibited only at large concentrations (100 μM). NMDA receptors were inhibited in a concentration-dependent manner. Tramadol and M1 metabolite inhibited the glutamate-concentration response curve without changing the half-maximal effective concentration or the Hill coefficient, indicating a noncompetitive inhibition. This study suggests that glycine receptors do not provide the antinociceptive effect of tramadol and that the inhibition of GABAA receptors at large concentration might correlate with convulsions. The inhibitory effect on NMDA receptors may contribute to the antinociceptive effect of tramadol at relatively large concentrations.