N-(4-Hydroxyphenyl) retinamide (4-HPR) is a synthetic retinoid that has shown biological activity against several malignant tumors and minimal side effects in humans. To explore the mechanisms underlying the chemotherapeutic effects of 4-HPR in glioblastoma, we used two human glioblastoma T98G and U87MG cell lines. In situ methylene blue staining showed the morphological features of astrocytic differentiation in glioblastoma cells following exposure to 1 microM and 2 microM 4-HPR for a short duration (24 h). Astrocytic differentiation was associated with an increase in expression of glial fibrillary acidic protein (GFAP) and downregulation of telomerase. Wright staining and ApopTag assay indicated appearance of apoptotic features in glioblastoma cells following exposure to 1 microM and 2 microM 4-HPR for a long duration (72 h). We found that 4-HPR caused apoptosis with activation of caspase-8 and cleavage of Bid to truncated Bid (tBid). Besides, apoptosis was associated with alterations in expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins resulting in an increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c and Smac, downregulation of selective baculoviral inhibitor-of-apoptosis repeat containing (BIRC) molecules, an increase in intracellular free [Ca2+], and activation of calpain and caspase-3. Taken together, these results strongly suggested that 4-HPR could be used at low doses for induction of both differentiation and apoptosis in human glioblastoma cells.