Protein phosphorylation is a major mechanism for the regulation of synaptic transmission. Previous studies have shown that several serine/threonine kinases are involved in the induction of long-term depression (LTD) at excitatory synapses on a Purkinje neuron (PN) in the cerebellum. Here, we show that Src-family protein tyrosine kinases (SFKs) are involved in the regulation of the LTD induction. Intracellular application of c-Src suppressed LTD. We also show that application of a SFK-selective inhibitor PP2 recovered LTD from the suppression caused by the inhibition of mGluR1 activity. These results indicate that SFKs negatively regulate the LTD induction at excitatory synapses on a cerebellar PN.