The aim of the present study was to explore the functions of histone acetyltransferase binding to origin recog-nition complex(ORC)1 (HBO1) during tumor development and to screen for HBO1 inhibitors. The chromatin immuno-precipitation sequencing(ChIP-seq) data of HBO1 in the RKO human colon cancer cell line(GSE33007) were downloaded from the Gene Expression Omnibus(GEO) database. The reads were then mapped back to a reference genomehg19. The PCR duplicate reads were removed by using SAMtools software and the shift was calculated using SPP and MaSC software. The peak calling was carried out using MACS1.4.0 software. Furthermore, the inhibitors of HBO1 were screened out from the Specs database using Dock6.6 software. The binding sites of HBO1 were mainly distributed in the intergenic, intronic and 3'-end regions. Further analysis revealed that a total of 9,467target genes was identified around HBO1 binding sites in the RKO cell lines and those genes mainly participated in the cell cycle, biosynthetic process, as well as other processes. Finally, 5inhibitors with best binding affinity in the positively charged cavity of HBO1 were screened out: i)5-[(2-hydroxybenzylidene)amino] -2-(2‑{4‑[(2‑hydroxy-benzylidene)amino]-2-sulfonatophenyl}vinyl)benzenesulfonate, ii)3-[4-(3-bromo-4-{2-[4-(ethoxycarbonyl)anilino]-2-oxo-ethoxy}-5-methoxybenzylidene)‑3‑methyl‑5‑oxo -4,5-dihydro-1H-pyrazol-1-yl]benzoic acid, iii)4-(4-{3-iodo‑5‑ methoxy‑4-[2-(2-methoxyanilino)-2-oxoethoxy]benzylidene}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid, iv)5-chloro-1,3-bis{[3,5,6-trihydroxy-4-(octyloxy)tetrahydro-2H-pyran-2-yl]methyl}-1,3-dihydro-2H-benzimidazol-2-one and v)4-{[4-(tetradecylamino)-1-naphthyl]diazenyl}benzoic acid. As a whole, in this study, we identified the possible binding sites and biological functions of HBO1. The potential inhibitors of HBO1 were also screened, which prove to be helpful for the inhibition of HBO1 during tumor development.