Fatty acids (FAs) are important components of cell membranes and precursors of bioactive molecules. Deficiency of FA has been associated with development of diseases and tissue damage in animal models. FAs regulate several cell functions including insulin secretion. We investigated the effects of FA withdrawal in culture medium on insulin producing cell (RINm5F) metabolism, survival, and response to death stimuli. Cells were cultivated in lipid-reduced fetal bovine serum (LRS) medium for 24, 48 or 72 h. Cell proliferation was measured by BrdU incorporation and glucose metabolism by 14CO2 production. Production of reactive oxygen species (ROS) was determined using dichlorofluorescein diacetate assay. Cells were exposed to death inducers to evaluate the effect of FA deprivation on RINm5F cell survival and susceptibility to death induction. Loss of plasma membrane integrity, DNA fragmentation, phosphatidylserine externalization and activation of caspases were analyzed by flow cytometry. The removal of FAs from the culture medium increased the number of dead cells as assessed by loss of plasma membrane integrity and occurrence of DNA fragmentation. Cells cultured in LRS exhibited higher cell death rates as compared to those cultivated with conventional serum. Cultivation in LRS for 48 hours increased phosphatidylserine externalization, caspase activation, ROS production and glucose oxidation. FA deprivation induced RINm5F cell death through apoptosis and exacerbated the cell death response to death stimuli. Cell death was associated with changes in generation of ROS and glucose oxidation.