Glycine receptors (GlyRs) are transmitter‐gated channels that mediate fast inhibitory neurotransmission in the spinal cord and brain. The GlyR β subunit contains a putative tyrosine phosphorylation site whose functional role has not been determined. To examine if protein tyrosine kinases (PTKs) regulate the function of GlyRs, we analysed whole‐cell currents activated by applications of glycine to CA1 hippocampal neurons and spinal neurons. The role of a putative site for tyrosine phosphorylation at position 413 of the β subunit was examined using site‐directed mutagenesis and expression of recombinant (α₁β^Y413F) receptors in human embryonic kidney (HEK 293) cells. Lavendustin A, an inhibitor of PTKs, depressed glycine‐evoked currents (I_Gly) in CA1 neurons and spinal neurons by 31 % and 40 %, respectively. In contrast, the intracellular application of the exogenous tyrosine kinase, cSrc, enhanced I_Gly in CA1 neurons by 56 %. cSrc also accelerated GlyR desensitization and increased the potency of glycine 2‐fold (control EC₅₀= 143 μm; cSrc EC₅₀= 74 μm). Exogenous cSrc, applied intracellularly, upregulated heteromeric α₁β receptors but not homomeric α₁ receptors. Substitution mutation of the tyrosine to phenylalanine at position β‐413 prevented this enhancement. Furthermore, a selective inhibitor of the Src family kinases, PP2, down‐regulated wild‐type α₁β but not α₁β^Y413F receptors. Together, these findings indicate that GlyR function is upregulated by PTKs and this modulation is dependent on the tyrosine‐413 residue of the β subunit.