Conjugated Linoleic Acid (CLA), a polyunsaturated fatty acid, refers to a group of dienoic derivatives of linoleic acid that can be found in natural food sources, such as milk fat and the meat of ruminant animals. CLA has been shown to have anti-carcinogenic activity in many in vitro and in vivo studies. Previous studies have focused on the effects of dietary CLA on the prevention of tumour appearance, yet relatively little is known about the actual mechanism of CLA’s anti-cancer activity. The most lethal aspect of cancer is the ability of tumour cells to metastasise and form secondary tumours. The matrix metalloproteinases (MMPs), a multi-gene family of enzymes, which degrade components of the extracellular matrix (ECM) have been implicated as major role players in tumour invasion and metastasis. The aim of this study was to examine the effect of CLA on the proliferation, migration, invasion and MMP-9 expression of a murine mammary cancer cell line, 4T1, which is known to be highly metastatic in vivo. Cells were treated with CLA, which contains a mixture of various isomers, and with the purified predominant isomers present in CLA, 9c, 11 i( l8:2) and 10/,12c(l 8:2). Cytotoxicity of CLA was examined by varying the concentration and incubation time. Sublethal as well as lethal doses were determined. Interestingly, it appeared that the 10/,l2 e ( l8 :2 ) isomer had the most lethal effect. Flow cytometric analysis revealed deregulation of the cell cycle correlating with the observed cytotoxic effects of CLA, especially with the 10i,12c(18:2) CLA isomer. The effect of CLA and its isomers on apoptosis was also investigated. The results from this assay were inconclusive, although examination of the cell cycle histogram plots indicated that cell death due to CLA was not occurring through an apoptotic pathway as there was no sub-Go/Gi peak. Treatment of the cells with a sub-lethal dose of CLA and its isomers resulted in a reduction of the invasive activity of the 4T1 cell line with the 9c, 11 18:2) isomer having the greatest effect. CLA and its isomers also resulted in a reduction in the percentage migration of the cells, especially the 9c, 11/(18:2) isomer and the CLA mixture. Substrate zymography gels were used to detect MMP activity and showed that the 4T1 cells expressed significant amounts of murine MMP-9. Following treatment with CLA and its isomers, there was no reduction in MMP expression.