Understanding the intricate network of molecular interactions responsible for synaptic plasticity remains a significant challenge in neuroscience research. Matrix metalloproteinase-9 (MMP-9) has been repeatedly implicated in the plasticity of excitatory synapses, notably in proBDNF processing. Our current study extends these findings by elucidating MMP-9's crucial role in dendritic spine plasticity through its involvement in the insulin-like growth factor I receptor (IGFIR) signaling pathway. Specifically, we demonstrate that MMP-9 is essential for IGFIR activation, a key process in long-term potentiation (LTP) induction.To investigate this, we employed glutamate uncaging in organotypic hippocampal slice cultures to induce long-term structural plasticity at individual synapses. Utilizing fluorescence lifetime imaging of an IGFIR FRET sensor, we examined IGFIR activity in the presence of MMP-9 inhibitors or in MMP-9 knockout (KO). Our findings reveal that MMP-9 inhibition or knockout significantly impairs IGFIR activation during LTP induction. Moreover, this effect can be rescued with MMP-9 overexpression in KO neurons. Further analysis identified insulin-like growth factor binding protein 2 (IGFBP2) as a potential MMP-9 substrate, suggesting a molecular mechanism through which MMP-9 modulates IGFIR signaling.Our results underscore MMP-9's pivotal role in dendritic spine plasticity at the onset of LTP induction through IGF-I signaling, highlighting its broader significance in neurotrophic factors processing on dendritic spines.