Lysophosphatidic acid (LPA) is an extracellular biological lipid and interacts with six subtypes of G protein-coupled LPA receptors (LPA(1) to LPA(6)). LPA receptors exhibit a variety of cellular functions, depending on types of cancer cells. In this study, to assess the roles of LPA(4) and LPA(6) in cell growth and motile activities of colon cancer cells, LPA(4) and LPA(6) knockdown cells were established from DLD1 and HCT116 cells. LPA treatment increased the cell growth activities of LPA(4) and LPA(6) knockdown cells, compared with control cells. The cell motile activities of LPA(4) and LPA(6) knockdown cells were significantly higher than those of control cells. To evaluate the effects of LPA(4) and LPA(6) on cell motile activity induced by anticancer drug, long-term fluorouracil (5-FU) treated (DLD-5FU) cells were generated. The expression levels of LPAR1, LPAR4 and LPAR6 genes were significantly increased in DLD-5FU cells. DLD5FU cells showed the high cell motile activity, compared with DLD1 cells. The increased cell motile activity was markedly stimulated by LPA(4) and LPA(6) knockdown. In contrast, the cell motile activity enhanced by 5-FU treatment was suppressed by LPA(1) knockdown. These results suggest that LPA signaling via LPA(4) and LPA(6) negatively regulates the cell motile activities of DLD1 and HCT116 cells as well as long-term 5-FU treated cells. (C) 2016 Elsevier Inc. All rights reserved.