CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene. It predominantly affects females that typically present with severe epileptic encephalopathy, intellectual disability, autistic features and motor dysfunction. Currently, there is no therapy apart from anti-epileptic drugs providing poor seizure management. To develop a gene replacement therapy, we initially characterised the CDKL5 isoforms expressed in human brain, neuronal cell lines, primary astrocytes and hESC-derived cortical interneurons, in which hCDKL5_1 and to a lesser extent hCDKL5_2 isoforms were found ubiquitously expressed. We cloned their coding regions downstream of the CBh promoter into ssAAV2 vector genome and produced high-titre rAAV vectors: AAV9, AAV-DJ for in vitro and AAV-PHP.B for in vivo applications. AAV-DJ vectors efficiently transduced CDKL5-mutant iPSC-derived neural progenitors, which were subsequently differentiated into neurons. hCDKL5_1 expression in CDKL5-mutant neurons led to an increased density of synaptic puncta, whilst hCDKL5_2 ameliorated the calcium signalling defect, implying distinct functions of these isoforms in neurons. Intrajugular delivery of AAV-PHP.B-GFP vectors in WT mice transduced neurons and astrocytes throughout the brain more efficiently than AAV9, and transduced spinal cord, DRGs, kidney and retina. Cdkl5 KO mice exhibited deficits in motor and cognitive behaviours compared to WT. Cdkl5 KO mice treated with AAV-PHP.B-hCDKL5_1 via intrajugular injection at age 28-30 days exhibited significant behavioural improvements compared to GFP-treated controls. Brain expression of hCDKL5_1 was more abundant in the hindbrain than the forebrain and midbrain. At the cellular level, transgene expression was sporadic in the brain and most prominent in CA1 hippocampal neurons and cerebellar Purkinje cells. Correction of PSD95 cerebellar misexpression, a major fine cerebellar structural abnormality in Cdkl5 KO mice, was found in regions of high transgene expression within cerebellum. This study provides the first evidence that AAV-mediated gene therapy can be effective in treating CDKL5 disorder.