Lafora disease is a rare and fatal form of progressive myoclonic epilepsy typically occurring early in adolescence. Common symptoms include seizures, dementia, and a progressive neurological decline leading to death within 5-15 years from onset. The disease results from mutations transmitted with autosomal recessive inheritance in the EPM2A gene, encoding laforin, a dual-specificity phosphatase, or the EPM2B gene, encoding malin, an E3-ubiquitin ligase. Laforin has glucan phosphatase activity, is an adapter of enzymes involved in glycogen metabolism, is involved in endoplasmic reticulum-stress and protein clearance, and acts as a tumor suppressor protein. Laforin and malin work together in a complex to control glycogen synthesis and prevent the toxicity produced by misfolded proteins via the ubiquitin-proteasome system. Disruptions in either protein can lead to alterations in this complex, leading to the formation of Lafora bodies that contain abnormal, insoluble, and hyperphosphorylated forms of glycogen called polyglucosans. We used the Epm2a -/- knock-out mouse model of Lafora disease to apply a gene replacement therapy by administering intracerebroventricular injections of a recombinant adeno-associated virus carrying the human EPM2A gene. We evaluated the effects of this treatment by means of neuropathological studies, behavioral tests, video-electroencephalography recording, and proteomic/phosphoproteomic analysis. Gene therapy with recombinant adeno-associated virus containing the EPM2A gene ameliorated neurological and histopathological alterations, reduced epileptic activity and neuronal hyperexcitability, and decreased the formation of Lafora bodies. Differential quantitative proteomics and phosphoproteomics revealed beneficial changes in various molecular pathways altered in Lafora disease. Improvements were observed for up to nine months following a single intracerebroventricular injection. In conclusion, gene replacement therapy with human EPM2A gene in the Epm2a -/- knock-out mice shows promise as a potential treatment for Lafora disease.