Highlights • CDKL5 deficiency disrupts the synaptic organization of thalamo-cortical (TC) and cortico-cortical (CC) connections in the barrel cortex (BC) • CDKL5 deficiency leads to BC hypoactivation • CDKL5 deficiency causes atypical whisker-mediated behavioural responses • CDKL5 deficiency does not prevent TC circuitry to undergo experience-dependent structural plasticity • Enhanced sensory stimulation restores cortical connectivity, BC activation levels and whisker-related behavioural responses ABSTRACT Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene cause CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental syndrome where patients exhibit early-onset seizures, intellectual disability, stereotypies, limited or absent speech, autism-like symptoms and sensory impairments. Mounting evidences indicate that disrupted sensory perception and processing represent core signs also in mouse models of CDD, however we have very limited knowledge on their underlying causes. In this study, we investigated how CDKL5 deficiency affects synaptic organization and experience-dependent plasticity in the thalamo-cortical (TC) pathway carrying whisker-related tactile information to the barrel cortex (BC). By using synapse-specific antibodies and confocal microscopy, we found that Cdkl5-KO mice display a lower density of TC synapses in the BC that was paralleled by a reduction of cortico-cortical (CC) connections compared to wild-type mice. These synaptic defects were accompanied by reduced BC activation, as shown by a robust decrease of c-fos immunostaining, and atypical behavioural responses to whisker-mediated tactile stimulation. Notably, a two-day paradigm of enriched whisker stimulation rescued both number and configuration of TC and CC synapses in Cdkl5-KO mice, and restored cortical activity as well as behavioural responses to control levels. Our findings disclose an important role of CDKL5 in controlling the organization and experience-induced modifications of excitatory connections in the BC and indicate how mutations of CDKL5 produce failures in higher-order processing of somatosensory stimuli.