Efforts to develop treatments for diseases caused by nonsense mutations have focused on identification of small molecules that promote translational read-through of messenger RNAs (mRNAs) harboring nonsense stop codons to produce full-length proteins. However, to date, no small molecule read-through drug has received FDA approval, probably because of a lack of balance between efficacy and safety. Depletion of translation termination factors eukaryotic release factor (eRF) 1 and eRF3a in cells was shown to promote translational read-through of a luciferase reporter gene harboring a nonsense mutation. In this study, we identified antisense oligonucleotides (ASOs) targeting translation termination factors and determined if ASO-mediated depletion of these factors could be a potentially effective and safe therapeutic approach for diseases caused by nonsense mutations. We found that ASO-mediated reduction of either eRF1 or eRF3a to 30%–40% of normal levels in the mouse liver is well tolerated. Hemophilia mice that express a mutant allele of human coagulation factor IX (FIX) containing nonsense mutation R338X were treated with eRF1- or eRF3a-ASO. We found that although eRF1- or eRF3a-ASO alone only elicited a moderate read-through effect on hFIX-R338X mRNA, both worked in synergy with geneticin, a small molecule read-through drug, demonstrating significantly increased production of functional full-length hFIX protein to levels that would rescue disease phenotypes in these mice. Overall our results indicate that modulating the translation termination pathway in the liver by ASOs may provide a novel approach to improving the efficacy of small molecule read-through drugs to treat human genetic diseases caused by nonsense mutations.