Imatinib is a highly selective tyrosine kinase inhibitor of the oncogenic kinase Bcr-Abl, the result of a chromosomal abnormality that is associated with chronic myeloid leukaemia (CML). Despite the success of this target-directed therapy, imatinib resistance is an emerging problem, especially in advanced stages of CML. In this study, we explored the yeast Saccharomyces cerevisiae as a model eukaryotic system to better understand the mode of action of imatinib, as well as potential mechanisms of resistance to this drug. Using a systematic approach, we screened a yeast haploid deletion collection with individual knockouts of most nonessential yeast genes, and identified 51 genes that are required for yeast resistance to imatinib. The genes identified are involved mainly in DNA repair and transcription control, cell cycle control and differentiation, vacuolar pH homeostasis, vesicular transport, and protein trafficking. Remarkably, approximately 80% of the genes identified in our screen have human orthologs. The vacuolar pH homeostasis function is associated to our dataset by 13 genes that encode subunits and assembly factors of the yeast vacuolar proton-translocating ATPase (V-ATPase). Further studies using fluorescence microscopy showed that physiological acidification of the vacuole is severely compromised following imatinib treatment of yeast cells, an effect that was found to be dose dependent. Results suggest that imatinib might act as an effective inhibitor of V-ATPase function in yeast, identifying V-ATPase activity and vacuolar function as novel imatinib targets.