Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to mutations of the AGXT gene encoding the peroxisomal enzyme alanine: glyoxylate-aminotransferase (AGT) which catalyzes the conversion of glyoxylate to glycine. In PH1 patients, glyoxylate cannot be efficiently converted into glycine and is instead oxidized to oxalate resulting in systemic oxalosis with deposition of insoluble calcium oxalate in kidneys and in other tissues, leading to nephrolithiasis, nephrocalcinosis, kidney failure, and systemic tissue damage. Combined liver/kidney transplantation is the only therapeutic strategy available to prevent disease progression. We hypothesize that overexpression of specific genes encoding enzymes involved in glyoxalate metabolism will steer glyoxylate towards alternative pathways to diminish oxalate production. To test this hypothesis, we overexpressed murine glyoxylate reductase/hydroxypyruvate reductase (GRHPR), that converts glyoxylate into glycolate, by a helper-dependent adenoviral vector (HDAd-GRHPR) in livers of Agxt−/− mice. The intravenous injection of HDAd-GRHPR resulted in significant reduction of hyperoxaluria and concomitant increase of serum glycolate that was not associated with signs of toxicity. Glutamate-pyruvate transaminase (GPT) in the cytosol transaminate glyoxylate using glutamate and alanine as amino-group donors. We hypothesize that GPT overexpression will steer glyoxylate towards transamination to diminish oxalate production. The intravenous injection of a helper-dependent adenoviral vector expressing murine GPT (HDAd-GPT) in Agxt−/− mice also resulted in significant and sustained reduction of hyperoxaluria. Interestingly, co-administration of both HDAd-GRHPR and HDAd-GPT resulted in further reduction and normalization of hyperoxaluria. Glycolate is one of the substrates leading to glyoxylate production, via peroxisomal glycolate oxidase (GO). We also show that an HDAd expressing a short hairpin RNA against GO resulted in reduction of hyperoxaluria in Agxt−/− mice. In summary, the results of this study show that metabolic diversion towards non-toxic metabolites has potential for treatment of PH1 and potentially other forms of hyperoxalurias, both primary and secondary. The metabolic diversion could be also obtained by RNA-based molecules expressing GRHPR and/or GPT or inhibiting GO activity. In addition, this study shows that HDAd vectors can be used to functionally validate therapeutic enzyme targets in inherited metabolic diseases.