Docosahexaenoic acid (DHA), the main member of the omega-3 essential fatty acid family, has been shown to reduce the invasion of the triple-negative breast cancer cell line MDA-MB-231, but the mechanism involved remains unclear. In the present study, a proteomic approach was used to define changes in protein expression induced by DHA. Proteins from crude membrane preparations of MDA-MB-231 cells treated with 100 µM DHA were separated by two-dimensional electrophoresis (2-DE) and differentially expressed proteins were identified using MALDI-TOF mass spectrometry. The main changes observed were the upregulation of Keratin, type Ⅱ cytoskeletal 1 (KRT1), catalase and lamin-A/C. Immunocytochemistry analyses confirmed the increase in KRT1 induced by DHA. Furthermore, in vitro invasion assays showed that siRNA against KRT1 was able to reverse the DHA-induced inhibition of breast cancer cell invasion. In conclusion, KRT1 is involved in the anti-invasive activity of DHA in breast cancer cells.