The aim of the current study was to analyze the differential microRNA (miRNA) expression profiles of human epidermal stem cells (ESCs) and differentiated keratinocytes. Enzyme digestion was used in combination with rapid adhesion to collagen IV to isolate primary human ESCs and differentiated keratinocytes, from which total RNA was extracted. Fluorescence labeling, microarray hybridization and differential expression analyses were performed. Reverse transcription quantitative polymerase chain reaction (RT‑qPCR) was performed to validate the reliability of the microarray results and predict the target genes of the differentially expressed miRNAs. A total of 25 miRNAs, including hsa‑miR‑197‑5p, hsa‑miR‑125b‑5p and hsa‑miR‑376a‑3p, were upregulated, whereas 166 miRNAs, including hsa‑miR‑29b‑3p, hsa‑miR‑203 and hsa‑miR‑34a‑3p, were downregulated in the human ESCs compared with the expression in differentiated keratinocytes. RT‑qPCR results confirmed the upregulation of hsa‑miR‑197‑5p and the downregulation of hsa‑miR‑29b‑3p, which were consistent with the microarray results. miRNA target prediction indicated that the miRNA expression levels correlated with cell proliferation, differentiation, apoptosis and senescence. Expression levels of miRNAs significantly differed between human ESCs and differentiated keratinocytes. This finding may be attributed to their biological characteristics, such as proliferative behavior and differentiation abilities.