Triacylglycerol (TG) accumulation caused by adipose triglyceride lipase (ATGL) deficiency or very low-density lipoprotein (VLDL) loading of wild-type (Wt) macrophages results in mitochondrial-mediated apoptosis. This phenotype is correlated to depletion of Ca 2+ from the endoplasmic reticulum (ER), an event known to induce the unfolded protein response (UPR). Here, we show that ER stress in TG-rich macrophages activates the UPR, resulting in increased abundance of the chaperone GRP78/BiP, the induction of pancreatic ER kinase-like ER kinase, phosphorylation and activation of eukaryotic translation initiation factor 2A, the translocation of activating transcription factor (ATF) 4 and ATF6 to the nucleus and the induction of the cell death executor CCAAT/enhancer-binding protein homologous protein. C16: 0 ceramide concentrations were increased in Atgl–/–and VLDL-loaded Wt macrophages. Overexpression of ceramide synthases was sufficient to induce mitochondrial apoptosis in Wt macrophages. In accordance, inhibition of ceramide synthases in Atgl–/–macrophages by fumonisin B1 (FB1) resulted in specific inhibition of C16: 0 ceramide, whereas intracellular TG concentrations remained high. Although the UPR was still activated in Atgl–/–macrophages, FB1 treatment rescued Atgl–/–macrophages from mitochondrial dysfunction and programmed cell death. We conclude that C16: 0 ceramide elicits apoptosis in Atgl–/–macrophages by activation of the mitochondrial apoptosis pathway.