The yeast Saccharomyces cerevisiae is a simple eukaryote and an excellent model organism for molecular biology. In this study, a NaCl-resistant S. cerevisiae mutant obtained by inverse metabolic engineering was used as a model to investigate responses and resistance behavior to NaCl, LiCl, KCl, TMA, spermine and sorbitol stresses., at physiological and genetic levels. The physiological spot test results revealed that the NaCl-resistant yeast mutant showed higher resistance to LiCl and NaCl. Gene expression analysis by qRT-PCR revealed that ENA6 and NHA1 genes of the mutant were induced in the absence and presence of LiCl and NaCl. The dysfunction of Na+/H+ antiporters are related to several diseases such as hypertension, epilepsy, postischemic myocardial arrhythmia, gastric and kidney disease, diarrhea, and glaucoma. Thus, the NaCl-resistant yeast mutant could be used to understand cellular sodium and lithium resistance mechanisms and the function of Na+/H+ antiporters also in higher eukaryotic organisms, including humans.