Chlorpromazine (CPZ) is a small permeable cationic amphiphilic molecule that inserts into membrane bilayers and binds to anionic lipids such as poly-phosphoinositides (PIs). Since PIs play important roles in many cellular processes, including signaling and membrane trafficking pathways, it has been proposed that CPZ affects cellular growth functions by preventing the recruitment of proteins with specific PI-binding domains. In this study, we have investigated the biological effects of CPZ in the yeast Saccharomyces cerevisiae. We screened a collection of approximately 4,800 gene knockout mutants, and found that mutants defective in membrane trafficking between the late-Golgi and endosomal compartments are highly sensitive to CPZ. Microscopy and transport analyses revealed that CPZ affects membrane structure of organelles, blocks membrane transport and activates the unfolded protein response (UPR). In addition, CPZ-treatment induces phosphorylation of the translation initiation factor (eIF2alpha), which reduces the general rate of protein synthesis and stimulates the production of Gcn4p, a major transcription factor that is activated in response to environmental stresses. Altogether, our results reveal that membrane stress within the cells rapidly activates an important gene expression program, which is followed by a general inhibition of protein synthesis. Remarkably, the increase of phosphorylated eIF2alpha and protein synthesis inhibition were also detected in CPZ-treated NIH-3T3 fibroblasts, suggesting the existence of a conserved mechanism of translational regulation that operates during a membrane stress.