Transcription factor Sp1 has recently been shown to be overexpressed in a number of human cancers and its overexpression contributes to malignant transformation. Sp1 regulates the expression of a number of genes participating in multiple aspects of tumorigenesis such as angiogenesis, cell growth and apoptosis resistance. To better understand the role of increased Sp1 levels on apoptosis regulation we have used retroviruses to overexpress this protein in haematopoietic Baf-3 cells and in 3T3 fibroblasts. We have also used inducible expression systems to control ectopic Sp1 levels in different cell types. Surprisingly, Sp1 overexpression on its own induces apoptosis in all the cellular models tested. The apoptotic pathways induced by Sp1 overexpression are cell type specific. Finally, using a truncated form of Sp1, we show that Sp1-induced apoptosis requires its DNA-binding domain. Our results highlight that Sp1 levels in untransformed cells must be tightly regulated as Sp1 overexpression leads to the induction of apoptosis. Our results also suggest that cancer cells overexpressing Sp1 can avoid Sp1-induced apoptosis.