N1,N11-diethylnorspermine (DENSPM), a polyamine analog that induces expression of spermidine/spermine N1-acetyltransferase (SSAT) and reduces polyamine levels in eukaryotic cells, has demonstrated anticancer effects in many cancer cell types. Gene expression of SSAT after treatment with DENSPM was measured in both U87 and LN229 cells using real-time PCR. Induction of SSAT mRNA using DENSPM resulted in significantly higher levels in U87 cells than in LN229 cells. Furthermore, DENSPM caused marked cell detachment in U87 cells and to a lesser extent in LN229 cells. We hypothesized that elevated SSAT expression plays a key role in DENSPM-induced cell detachment in glioblastoma cells. To investigate whether forced expression of SSAT would lead to reduced cell adhesion and increased cell detachment, we transfected a PCMV-SSAT plasmid into LN229 cells and observed significant cell detachment. In addition, we treated U87 cells with SSAT siRNA together with DENSPM to blunt the induction of SSAT by DENSPM. This resulted in an inhibition of cell detachment in U87 cells compared with the DENSPM treatment alone. Increased SSAT expression by transfection enhanced the DENSPM cell-kill effect in LN229 cells whereas reduction of SSAT by siRNA attenuated the DENSPM cell-kill effect. The protein levels of AKT, mTOR and integrin α5β1, which are members of the cell adhesion and anti-apoptotic signal transduction pathways, were decreased in the PCMV-SSAT transfected LN229 cells. Collectively, these results demonstrate that SSAT induction at least partially plays a role in cell detachment and apoptosis of glioblastoma cells by DENSPM treatment.