Calcium-evoked exocytosis of synaptic vesicles at the presynapse involves recruitment, docking, and priming of synaptic vesicles as well as localization of voltage-gated calcium channels in their close proximity. Piccolo, a large multidomain protein, acts as a scaffolding protein at the active zone, thus mediating the exact localization and functional interaction of several involved proteins. We aimed to investigate the functional role of piccolo, namely the effect on glutamate release and the involvement of calcium channel regulation, using cortico-hippocampal cell cultures from a novel conditional Pclo knockout mouse model. Transduction with cre-expressing viral vectors resulted in complete loss of piccolo. We performed quantitative immunostaining with antibodies against specific presynaptic proteins and observed changes in components of the presynaptic release machinery as well as in proteins expressed in the synaptic vesicle membrane. Via live imaging of synaptic vesicle recycling using the genetically encoded pH sensor SypHymOR, we observed a reduced releasable pool of synaptic vesicles, which is in line with the previously published role of piccolo in recycling of synaptic vesicles. Nevertheless, live imaging of evoked glutamate release with the genetically encoded glutamate sensor SF-iGluSnFR.S72A at single-synapse level revealed that loss of piccolo leads to a reduced glutamate release and an altered short-term plasticity assessed as paired-pulse facilitation. Importantly, calcium dependence of release and paired-pulse facilitation was dramatically reduced, while presynaptic calcium imaging with the genetically encoded calcium indicator Syn-GCamp6f confirmed unaltered evoked calcium influx. These data indicate that piccolo is required for normal calcium sensitivity of glutamate release in cortico-hippocampal synapses.