Targeted ectopic expression of interferon-gamma (IFN-gamma) in the eyes of transgenic mice disrupts lens differentiation, upregulates immunoproteasomes, and causes cataract. In this study, the hypothesis that IFN-gamma induces proteasome-dependent apoptosis of lens epithelial cells was tested. Murine lens epithelial alphaTN4-1 cells were treated with IFN-gamma. Apoptosis was measured using annexin V-FITC and propidium iodide (PI) staining, and DNA fragmentation. IFN-gamma-inducible mRNA and protein expressions were measured by RT-PCR and Western blot analysis. Caspase activities were measured using colorimetric substrates and poly (ADP ribose) polymerase (PARP) cleavage. The effect of proteasome inhibition was tested with MG132 and lactacystin. IFN-gamma treatment at a concentration that induces immunoproteasome expression causes an approximately 20% increase in early apoptotic cells as observed by annexin V-FITC/PI staining and the increase in DNA fragmentation. IFN-gamma-induced apoptosis was accompanied by upregulation of apoptosis-related genes, including a dramatic increase in signal transducer and activator of transcription (STAT)-1 and interferon consensus sequence binding protein (ICSBP), a more than 2-fold increase in IRF-1, and a 1.7- to 2-fold increase in caspase-1 mRNA. Bcl-2 mRNA decreased 2.4- to 3.0-fold, whereas Bax mRNA was unchanged. The Bax-to-Bcl-2 protein ratio increased by 1.6-fold. Caspase-1 and -8 activities were higher, but there was no increase in caspase-3 activity. Proteasome inhibitors MG132 and lactacystin protected the cells against IFN-gamma-induced apoptosis. A positive control treatment with staurosporine (STP) caused increased caspase-3 activity, which was inhibited by MG132. IFN-gamma causes apoptosis of alphaTN4-1 cells, accompanied by upregulation of known effectors. IFN-gamma-induced apoptosis involves Bcl-2 family proteins and caspases. Proteasome inhibition has antiapoptotic effects on IFN-gamma-induced apoptosis. It also inhibits the STP-induced increase in caspase-3 activity. If IFN-gamma-induced apoptosis of lens epithelial cells contributes to cataractogenesis, the proteasome may be a therapeutic target.