Hyperforin (Hyp), the major lipophilic constituent of Hypericum perforatum (St John’s wort), was assayed as a stable dicyclohexylammonium salt (Hyp-DCHA) for cytotoxicity and inhibition of matrix proteinases, tumour invasion and metastasis. Hyp-DCHA triggered apoptosis-associated cytotoxic effect in both murine (C-26, B16-LU8 and TRAMP-C1) and human (HT-1080 and SK-N-BE) tumour cells; its effect varied, with B16-LU8, HT-1080 and C-26 the most sensitive (IC50 = 5 to 8 μmol/l). At these concentrations, a marked and progressive decline of growth was observed in HT-1080 cells, whereas untransformed endothelial cells were only marginally affected. Hyp-DCHA inhibited in a dose-dependent and non-competitive manner various proteinases instrumental to extracellular matrix degradation; the activity of leukocyte elastase was inhibited the most (IC50 = 3 μmol/l), followed by cathepsin G and urokinase-type plasminogen activator, whereas that of the matrix metalloproteinases (MMPs) 2 and 9 showed an IC50>100 μmol/l. Nevertheless, inhibition of extracellular signal-regulated kinase 1/2 constitutive activity and reduction of MMP-2 and MMP-9 secretion was triggered by 0.5 μmol/l Hyp-DCHA to various degrees in different cell lines, the most in C-26. Inhibition of C-26 and HT-1080 cell chemoinvasion (80 and 54%, respectively) through reconstituted basement membrane was observed at these doses. Finally, in mice that received IV injections of C-26 or B16-LU8 cells, daily intra-peritoneal administration of Hyp-DCHA – without reaching tumour-cytotoxic blood levels – remarkably reduced inflammatory infiltration, neovascularisation, lung weight (−48%) and size of experimental metastases with C-26 (−38%) and number of lung metastases with B16-LU8 (−22%), with preservation of apparently healthy and active behaviour.