The thiazolidinedione (TZD) class of peroxisome proliferator-activated receptor (PPAR) γ ligands, known for their ability to induce adipocyte differentiation and increase insulin sensitivity, also exhibits anticancer properties. Currently, TZDs are being tested in clinical trials for treatment of human cancers expressing high levels of PPARγ because it is assumed that activation of PPARγ mediates their anticancer activity. Using PPARγ^−/− and PPARγ^+/+ mouse embryonic stem cells, we report here that inhibition of cell proliferation and tumor growth by TZDs is independent of PPARγ. Our studies demonstrate that these compounds block G₁-S transition by inhibiting translation initiation. Inhibition of translation initiation is the consequence of partial depletion of intracellular calcium stores and the resulting activation of protein kinase R that phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2), thus rendering eIF2 inactive. PPARγ-independent inhibition of translation initiation most likely accounts for the anticancer properties of thiazolidinediones.