Objective To establish effective chromatin immnuoprecipitation (ChIP) and identify the interaction between the chromatin DNA of MDA-MB-231 cells and DNA methyltransferase 3b (DNMT3b), histone deacetylase 1(HDAC1) protein by ChIP techique. Methods The condition of ChIP assay was established and optimized. The MDA-MB-231 cells were treated with either 5-aza-2′-deoxycytidine (5-aza-dc), trichostatin A (TSA) seperately or the combination of the two drugs. Then DNMT3b and HDAC1 binding to SLC22A18 gene promoter region were detected by ChIP assay using specific DNMT3b and HDAC1 antibodies. PCR amplified its gene specific DNA fragemnt. Western blotting was used to examine protein levels of DNMT3b and HDAC1. Results SLC22A18 gene specific fragments were detected in the DNA fragments immunoprecipitated by DNMT3b and HDAC1 antibodies. DNMT3b was dissociated from the SLC22A18 promoter after treatment of MDA-MB-231 cells with 5-aza-dc alone or in combination with TSA. Treatment of MDA-MB-231 cells with TSA or in combination with 5-aza-dc reduced the association of HDAC1 with SLC22A18 promoter. The DNMT3b and HDAC1 protein levels were unaffected by either 5-aza-dc, TSA alone or combined together. Conclusion The results confirm that DNMT3b and HDAC1 was binded to SLC22A18 promoter in MDA-MB-231 cells and were involved in transcriptional regulation of SLC22A18 gene.