Evidence indicates that diets enriched in Docosahexaenoic acid (DHA), a 22:6 n-3 polyunsaturated fatty acid, reduces the risk of prostate cancer, but the biochemical mechanisms are unclear. The Hippo pathway has been well established as a tumor suppressor pathway and is involved in many diverse biologic processes including cell growth, cell death, and organ size control in organisms. Here we showed that DHA induces cell growth inhibition and apoptosis of human androgen-independent prostate cancer cells dependent on the Hippo pathway. DHA inactivates YAP by promoting phosphorylation in androgen-independent prostate cancer cell lines, accompanied by increased YAP cytoplasm translocation. We also observed that DHA-induced YAP phosphorylation was reversed by both the LATS1 and MST1 siRNAs. Further experiments showed that the mechanism of DHA-induced YAP phosphorylation associated with FFAR1 and FFAR4. Down-regulation of FFAR1 and FFAR4 resulted in reduced YAP phosphorylation and reversed DHA-induced YAP phosphorylation. In addition, DHA-induced YAP phosphorylation was abolished by dominant negative Gαs and PKA inhibitor H-89. Overall, these findings define a mechanism by which FFAR1-and FFAR4-dependent activation of Hippo pathway mediates DHA-induced apoptosis of androgen-independent prostate cancer cells, thus providing a promising therapeutic target for prostate cancer.