This study investigates the effects of omega-3 fatty acid, docosahexaenoic acid (DHA-22:6 n-3), a constituent of fish oil, on the cytotoxicity of docetaxel in prostate cancer cells. Materials and methods: Cell viability was assessed using the MTT assay and apoptosis was evaluated by flow cytometry using PI, annexin V, and JC-1 staining. Cellular signalling mechanisms that might explain the observed pro-apoptotic effects were investigated using NF-κB pathway specific cDNA microarrays and subsequent RT-PCR validation of candidate genes. Results: DHA enhanced the pro-apoptotic effect of docetaxel, synergistically, in hormone dependent and independent LNCaP, DU145 and PC3 cells, respectively. Cell cycle study showed an increase in G2M arrest and JC-1 staining showed a significant (p<0.05) increase in apoptosis with combination treatments in LCNaP cells. Molecular studies using the NF-κB pathways specific microarrays and validation by RTPCR showed decreased expression of FADD, AKT1, MAX, TRAF3, MAP2k4, TNFRSF11A and RIPK1 in LNCaP cells. Similar results were obtained with DU145 cells. Combination treatment was more effective than single treatments. Combination treatments suppressed the gene involved in NF-κB signalling, that was induced by docetaxel alone due to stress. Conclusion: DHA synergistically potentiated by cytotoxic effect of docetaxel in prostate cancer cells through increased apoptosis by modulation of the genes associated with NF-κB pathway. This supports the possibility of developing such combination modalities for treatment to enhance the effect of chemotherapy in prostate cancer.