Fabry disease is one of the most common lysosomal storage disorders that results from a deficiency of the enzyme alpha galactosidase A (GLA). The deficiency leads to systemic accumulation of globotriaosylceramide (Gb3) and its deacylated derivative, Lyso-Gb3, in multiple organs, which can progress into end stage renal disease, heart failure or stroke in Fabry patients. Despite current FDA-approved enzyme replacement and chaperone therapies, unmet medical needs remain. A4GALT is the key enzyme that catalyzes the incorporation of galactose to lactosylceramide to form Gb3. We tested the hypothesis whether a substrate reduction approach utilizing an antisense oligonucleotide (ASO) targeting A4GALT can ameliorate Lyso-Gb3 accumulation in a mouse model of Fabry disease, the α-Gal A knockout (GLA KO). Potent and well tolerated mouse A4GALT ASO leads were identified in wildtype (WT) mice and evaluated in a dose response (5, 20, 40, 80 mg/kg/week) in GLA KO mice. A4GALT reductions of up to 90% were observed in the kidney. Lyso-Gb3 level was determined by LC-MS and was found to be reduced in a dose-dependent manner by up to 75% and 65% in plasma and kidney, respectively. In conclusion, A4GALT ASO treatment in GLA KO mice results in dose-dependent reductions of plasma and kidney lyso-Gb3, a key endpoints of disease efficacy that are monitored in the clinic. Hence, a substrate reduction therapy by inhibiting A4GALT with ASO could be a promising approach for treatment of Fabry disease.