Chromate is a widespread pollutant as a waste of human activities. However, the mechanisms underlying its high toxicity are not clearly understood. In this work, we used the yeast Saccharomyces cerevisiae to analyse the physiological effects of chromate exposure in a eukaryote cell model. We show that chromate causes a strong decrease of sulfate assimilation and sulfur metabolite pools suggesting that cells experience sulfur starvation. As a consequence, nearly all enzymes of the sulfur pathway are highly induced as well as enzymes of the sulfur-sparing response such as Pdc6, the sulfur-poor pyruvate decarboxylase. The induction of Pdc6 was regulated at the mRNA level and dependent upon Met32, a coactivator of Met4, the transcriptional activator of the sulfur pathway. Finally, we found that chromate enters the cells mainly through sulfate transporters and competitively inhibits sulfate uptake. Also consistent with a competition between the two substrates, sulfate supplementation relieves chromate toxicity. However, the data suggest that the chromate-mediated sulfur depletion is not simply due to this competitive uptake but would also be the consequence of competitive metabolism between the two compounds presumably at another step of the sulfur assimilation pathway.