Abstract: OBJECTIVE To design and synthesize a series of 4H-pyran-arylurea derivatives and to evaluate their anticancer activities in vitro. METHODS Nitro-containing pyran intermediates was synthesized by "one-pot" condensation of 3-nitrobenzaldehyde, malononitrile, dimethyl 3-oxopentanedioate. Reduction of the nitro group of above compound with iron powder, and then condensed with isocyanates to afford target compounds. Human large cell lung cancer cells H460, human lung cancer cells A549 and human colon cancer cells HT-29 cell lines were employed to evaluate anticancer activities of these compounds using MTT-based assay. RESULTS Eleven 4H-pyran derivatives bearing arylurea moieties were synthesized. The results of in vitro antiproliferative activity showed 11 compounds had good inhibitory activities against 3 tumor cell lines. Among them, compound 7c exhibited remarkable inhibitory activity against H460 and A549 cell lines with IC₅₀ value of 0.82 and 0.98 μmol·L^-1, respectively, which were more potent than that of the positive control sorafenib(IC₅₀=3.20 and 2.83 μmol·L^-1). CONCLUSION 4H-pyran derivatives bearing arylurea moieties show excellent anticancer activities, and they can be used as the structural skeleton of anticancer compounds for futher study.