Monocular deprivation-induced ocular dominance plasticity (MD-ODP) in the visual cortex is an established in vivo paradigm, in which inactivity-induced synaptic plasticity contributes to the functional reconfiguration of cortical networks. Bassoon (Bsn) is a presynaptic protein involved in the spatial and functional organisation of synaptic vesicle (SV) recycling. Here, we report a novel role for Bsn in the inactivity-induced functional reconfiguration of adult neocortical networks in vivo and in vitro. We subjected Bsn knock-out mice to MD and imaged visual stimulus-evoked intrinsic signals in primary visual cortex. While MD-ODP was unaffected in juveniles, adult Bsn KO mice failed to develop MD-ODP. Animals with Bsn deletion restricted to the neocortical excitatory synapses also failed to develop adult MD-ODP, whereas subcortical reflexes could be trained, further confining the specific site of Bsn action. In vitro experiments revealed a perturbed inactivity-induced upscaling of presynaptic neurotransmitter release due to aberrant inactivity-induced regulation of the readily releasable and recycling SV pools. Postsynaptic scaling, driven by an increase in AMPA receptor abundance, was unaffected. Confirming the importance of the modulation of SV pools during MD-ODP, we detected specific changes in the phosphorylation of synapsin, the main regulator of SV clustering, in visual cortices upon MD. Finally, we showed that MD did not affect the synapsin phosphorylation in neocortex-specific Bsn mutants that failed to develop MD-ODP. Our results reveal presynaptic homeostatic plasticity as an important mechanism contributing to the activity-dependent functional shaping of the neocortical circuity and Bsn as an indispensable molecular player in this process.