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Objective To investigate the in vitro anticancer effects of 4-ethoxy-2-hydroxy-6-methyl benzoic acid (D6B2) and its combinative anticancer effects with clinical anticancer drug cis-DDP. Methods Human cancer cell lines CNE, Hep G2, HeLa, QBC939 and K562 were used for the in vitro anticancer studies. The anticancer effects were detected by cell counting or MTT assay. Results The inhibitory ratios of D6B2 (25 µM, 50 µM, 100 µM, 150 µM, 200 µM) were 24.94%, 42.11%, 65.74%, 70.22%, 73.11% on CNE; 23.15%, 54.56%, 62.95%, 70.14%, 75.18% on Hep G2; 31.34%, 60.76%, 78.43%, 85.78%, 90.75% on HeLa; 5.97%, 12.05%, 28.44%, 35.28%, 51.31% on QBC939; 18.20%, 35.48%, 46.76%, 58.44%, 57.32% on K562. The IC50 value against CNE, HepG2, HeLa, QBC939 and K562 were 66.9 µM, 60.98 µM, 40.7 µM, 197.07 µM and 120.06 µM, respectively. The inhibitory ratios of D6B2 (25 µM, 50 µM, 100 µM, 150 µM, 200 µM) on HeLa were 7.96%, 9.56%, 10.98%, 14.00%, 17.33% at 24 h; 10.62%, 25.96%, 47.63%, 48.24%, 49.03% at 48 h; 18.08%, 53.68%, 85.55%, 87.86%, 82.07% at 72 h. The CDI value of D6B2 at 25 µM, 50 µM and 100 µM was 0.039, 0.019 and 0.014, respectively, when used with 10 µM DDP. Conclusion D6B2 showed significant in vitro anticancer effects on human cancer cells. Its in vitro anticancer activity was dose-dependent and time-dependent. D6B2 and DDP had synergistic in vitro anticancer effects. [ABSTRACT FROM AUTHOR]