Accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER) causes ER stress. Snf1, the Saccharomyces cerevisiae ortholog of AMP-activated protein kinase (AMPK), plays a crucial role in the response to various environmental stresses. However, the role of Snf1 in ER stress response remains poorly understood. In this study, we characterize Snf1 as a negative regulator of Hog1 MAPK in ER stress response. The snf1 mutant cells showed the ER stress resistant phenotype. In contrast, Snf1-hyperactivated cells were sensitive to ER stress. Activated Hog1 levels were increased by snf1 mutation, although Snf1 hyperactivation interfered with Hog1 activation. Ssk1, a specific activator of MAPKKK functioning upstream of Hog1, was induced by ER stress, and its induction was inhibited in a manner dependent on Snf1 activity. Furthermore, we show that the SSK1 promoter is important not only for Snf1-modulated regulation of Ssk1 expression, but also for Ssk1 function in conferring ER stress tolerance. Our data suggest that Snf1 downregulates ER stress response signal mediated by Hog1 through negatively regulating expression of its specific activator Ssk1 at the transcriptional level. We also find that snf1 mutation upregulates the unfolded protein response (UPR) pathway, whereas Snf1 hyperactivation downregulates the UPR activity. Thus, Snf1 plays pleiotropic roles in ER stress response by negatively regulating the Hog1 MAPK pathway and the UPR pathway.