Worldwide, multiple Fusarium mycotoxins occur as contaminants of cereals with important impacts on human and animal health. The aim of this study was to investigate the effects of the widespread Fusarium secondary metabolite enniatin (ENN), a cyclic hexadepsipeptide, on human cell growth and survival. While short-term exposure (up to 8 h) to ENN at nanomolar concentrations slightly but significantly stimulated cell proliferation, it showed profound apoptosis-inducing effects especially against various human cancer cell types at low micromolar concentrations (already after 24 h of treatment). Several cellular changes indicative for programmed cell death such as cell shrinkage, chromatin condensation, DNA fragmentation, and apoptotic body formation were observed. Correspondingly, the cleavage of poly(ADP-ribosyl)polymerase and the activation of multiple caspases accompanied a distinct loss of mitochondrial membrane potential. To investigate the impact of apoptosis- and cell cycle-regulating proteins on ENN activity, HCT116 cells with homozygously disrupted p53, p21, or bax genes were analyzed. In vitality assays, no significant influences of these proteins on the anticancer activity of ENN were detectable. In contrast, 3H-thymidine incorporation revealed a significantly more efficient block of DNA synthesis in p53 wild-type as compared to knock-out cells. Accordingly, fluorescence-activated cell sorting analysis demonstrated a stronger ENN-induced cell cycle arrest in the G0/G1 phase. Profound ENN-mediated induction of p53 and the p53-downstream cell cycle inhibitor p21 were detectable in p53 wild-type cells by Western blotting. P53-independent p21 induction was also detectable at higher ENN concentrations in p53 (-/-) cells. In contrast, bax activation by ENN was independent of the cellular p53 status. In summary, our results suggest that short-term exposure to very low ENN concentrations, for example, via food intake, might have tumor-promoting functions based on growth stimulation. In contrast, elevated ENN concentrations exert profound p53-dependent cytostatic and p53-independent cytotoxic activities especially against human cancer cells, suggesting a potential quality of ENN as an anticancer drug.