Cytarabine is an important medicine for acute myeloid leukemia (AML) treatment, however, drug resistance hinders the treatment of AML. Although microRNA (miRNA or miR) 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 the present 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. Differentially expressed mRNAs (DEMs) and differentially expressed genes (DEGs) with more than two-fold changes between HL60 and R-HL60 cells were screened out. Negatively associated miRNA-mRNA pairs were selected as candidate miRNA-mRNA target pairs according to the miRDB, Targetscan or miRTar databases. Functional enrichment analysis of DEGs included in the candidate miRNA-mRNA pairs was performed. The results indicated that 10 DEGs (
CCL2
,
SOX9
,
SLC8A1
,
ICAM1
,
CXCL10
,
SIPR2
,
FGFR1
,
OVOL2
,
MITF
and
CARD10)
were simultaneously involved in seven Gene Ontology pathways related to the regulation of migration ability, namely the ‘regulation of cell migration’, ‘regulation of locomotion’, ‘regulation of cellular component movement’, ‘cell migration’, ‘locomotion’, ‘cell motility’, and ‘localization of cell’. DEMs predicted to negatively regulate the aforementioned 10 DEGs were paired with DEGs into 16 candidate miRNA-mRNA pairs related to the regulation of migration ability. In addition, migration assays revealed that the migration ability of R-HL60 cells was greater than that of HL60 cells. These findings provide a new perspective for the treatment of cytarabine-resistant AML and advance our understanding of altered migration ability underlying cytarabine resistance development, specifically related to miRNAs.