〔Abstract〕 Objective To investigate the effects of DNA demethylation drugs combined with histone deacetylase inhibitors on fragile X mental retardation 1 neighbor protein(FMR1NB) expression and its promoter methylation in human oral cancer cells and try to find a strategy of weakening the heterogeneity of FMR1NB expression. Methods Human oral cancer cell lines Cal27 and SCC-9 were treated with decitabine(DAC), an inhibitor of DNA methyltransferase, combined with trichostatin A(TSA) and valproic acid(VPA), inhibitors of histone deacetylase. Then reverse transcription-polymerase chain reaction(RT-PCR), quantitative real-time PCR(qRT-PCR)and Western blot were used to detect the expression of FMR1NB and pyrosequencing was used to detect the methylation of FMR1NB promoter. Results Compared with the blank control group, DAC and its combination with TSA and VPA significantly induced the expression of FMR1NB mRNA and protein in Cal27 and SCC-9 cells. Compared with DAC alone group, FMR1NB mRNA expression of each DAC-combined drug groups significantly increased, but FMR1NB protein did not significantly change in Cal27 cells; for SCC-9 cells, except for DAC+TSA group, the mRNA and protein levels of FMR1NB significantly increased in all other groups. In addition, there was no significant difference in the expression of FMR1NB mRNA and protein between the three-combined drugs group and two-combined drugs groups. Further methylation assay showed that the methylation level of the overall FMR1NB promoter and its each CpG site measured were reduced to varying degrees in all treatment groups except for three-combination drug group of SCC-9. Conclusion DAC and its combination with TSA and VPA can enhance the expression of FMR1NB by mediating the demethylation of FMR1NB promoter, wherein the enhanced expression effect of the combination of the two drugs is stronger, suggesting that they have the potential to weaken the heterogeneity of FMR1NB expression and improve the immunotherapy effect of oral cancer.