In order to exert metabolic effects, biologically important fatty acids must be efficiently taken up by the cells. Therefore, the main aim of this investigation was to examine the uptake of fatty acids representing three different series such as oleic acid (OA, 18:1n-9), arachidonic acid (AA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5n-3) by MDA-MB-231 breast cancer cells. In addition, we investigated the effects of insulin and several adipocyte derived proteins (TNF-α, adiponectin and leptin) on the fatty acid uptake by these cells as obesity and insulin resistance syndrome have been suggested as one of the possible mechanisms by which obesity affects breast cancer risk. Cells were incubated with radiolabeled fatty acids for 3 h at concentrations of 50 and 100 µM, and the uptake was determined using Liquid scintillation counting (LSC). Protein concentrations were estimated by Bicinchoninic acid (BCA) assay. Data were expressed as nmol of fatty acid taken up/mg of cellular protein. Pre-stimulation of these cells with insulin and other factors was performed 24 h before the initiation of fatty acid uptake studies.We report for the first time that AA was predominantly taken up by these cells compared with EPA and OA. Our findings suggest that large amount of AA uptake may help these cancer cells to promote their growth and proliferation However, uptake of any of these fatty acids was not affected by the presence of insulin, TNF-α, adiponectin or leptin.