Human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause in vitro caspase-independent cell death, accompanied by previously undescribed cytoplasmic vacuolization with abnormal fusion of various types of endosomal-lysosomal organelles. Yeasts are unicellular eukaryotes widely used as a biological model to study fundamental cellular processes. In particular, yeast models of cell death are useful due to the similarity of regulated cell death mechanisms between yeast and humans. This, together with the functional similarity of yeast vacuoles and the lysosomal/endosomal compartment of mammalian cells, suggests yeasts to be a promising model to study molecular mechanisms of 3Cpro action. Here, we expressed 3Cpro and its mutant inactivated form (3Cmut) in Saccharomyces cerevisiae using pYES2 vector. It was shown that expression of 3Cpro, but not 3Cmut, caused cells to lose the ability to proliferate, but maintained viability, thus indicating the enzyme exerts a cytostatic effect. In addition, using vacuolar dyes and light and electron microscopy, it was found that 3Cpro causes vacuole distortion and fragmentation, accompanied by the abnormal vacuolar staining. In summary, the effects of the 3Cpro on yeast cells resemble the response of mammalian cells. Therefore, the experimental system based on S. cerevisiae can be used for further studies of 3Cpro action.