Ionotropic glycine receptors (GlyRs) are chloride-permeable ligand-gated ion channels expressed in neurons of the central nervous system. These receptors play a key role in regulating neuronal membrane potential, impacting various physiological processes, including motor control, respiratory rhythm, and pain modulation. Consistent with its relevance, positive allosteric modulators (PAMs) of GlyRs have shown analgesic effects in chronic pain models. A promising group of glycinergic PAMs are the tricyclic sulfonamides (TS), such as AM-1488, which has been majorly studied using biochemical and high-throughput techniques. Nevertheless, the biophysical and neurophysiological mechanisms underlying the GlyR modulation by TS have not been fully elucidated. Here, we examined the functional effects of AM-1488, a prototypical TS, on recombinant and native GlyRs by electrophysiology. Whole-cell recordings on recombinant GlyRs indicate that AM-1488 enhances glycine-evoked currents in both homo- and heteromeric α1, α2, and α3 GlyRs with similar potency and efficacy. Additionally, we observed a subunit selective direct activation of GlyRs by AM-1488 at concentrations of 3-10 µM. Analysis performed on spinal GlyRs reproduced the positive allosteric modulation and agonistic effects of AM-1488 encountered in recombinant systems. In a synaptic context, AM-1488 did not alter the frequency, the amplitude nor the kinetic parameters of glycinergic mIPSCs, but induce tonic-like currents. Altogether, our study contributes to comprehend the mechanisms of this class of PAMs and to begin our understanding of the neurophysiological origin of the glycinergic analgesia mediated by TS compounds.