ABSTRACT: Cytarabine is the main drug for acute myeloid leukemia (AML) treatment; however, drug resistance hinders the treatment of AML. Although microRNA (miRNA) alteration is one of the well-recognized mechanisms underlying drug resistance in AML, few studies have investigated the role and function of miRNAs in the development of cytarabine resistance. In this study, total RNA was isolated from parental HL60 and cytarabine resistant HL60 (R-HL60) cells. Subsequently, miRNAs and mRNAs were detected using small RNA sequencing and gene expression array, respectively. The miRNAs and genes with ≥ 2-fold difference in expression between HL60 and R-HL60 cells were screened out. Negatively correlated miRNA–mRNA pairs were selected as candidate miRNA–mRNA target pairs by using the miRDB, Targetscan or miRTar databases. Functional enrichment analysis of differentially expressed genes (DEGs) included in the candidate miRNA–mRNA network was performed. The results revealed that CCL2, SOX9, SLC8A1, ICAM1, CXCL10, SIPR2, FGFR1, OVOL2, MITF, and CARD10 were simultaneously involved in seven GO pathways, namely the regulation of cell migration, regulation of locomotion, regulation of cellular component movement, cell migration, locomotion, cell motility, localization of cell. These genes were negatively correlated with the altered miRNAs (miR-1-3p, miR-155-5p, miR-1255b-5p, miR-200c-5p, miR-3609, miR-1285-3p, miR-124-3p, miR-146a-5p, miR-497a-5p, and miR-3150a-5p), suggesting that they are the potential targets of the miRNAs to regulate cell migration behavior or ability. Therefore, our results advance our understanding of the regulatory mechanism underlying cytarabine resistance development, specifically related to miRNAs.