To study the expression of microRNA-544 (miR-544) in hepatocelluar carcinoma (HCC) tissues and its effect on proliferation, apoptosis, colony formation and sphere-forming potential of HCC cells, so as to explore the role of miR-544 in carcinogenesis and development of HCC. Methods Quantitative real-time PCR was used to detect the expressions of miR-544 in liver tissues from rats treated with diethylnitrosamine (DEN), in HCC and adjacent non-tumor tissues from patients, and in HCC spheres after sphere-forming culture. After HCC cell line Hep3B being transfected with miR-544 mimic or miR-544 inhibitor, cell counting kit-8 (CCK-8) was used to detect the proliferation of the HCC cells, colony-formation assay was used to observe the colony-formation ability of the HCC cells, and flow cytometry was used to analyze the cell apoptosis of HCC cells with propidium iodide staining. The sphere-formation assay was performed to determine the sphere-forming potential of HCC cells transfected with miR-544 mimic. Results MiR-544 expression was significantly down-regulated in the HCC tissues of DEN-treated rats (versus the control rats without modelling, P<0.05) and HCC tissues of patients (versus adjacent non-tumor tissues, P<0.01). Overexpression of miR-544 significantly inhibited proliferation (P<0.05, P<0.01) and colony-formation potential (P<0.05) of HCC cells, and also significantly promoted the apoptosis of HCC cells at 72 h (P<0.01). While inhibition of miR-544 in HCC cells significantly promoted the cell proliferation (P<0.01) and colony-formation potential (P<0.05). MiR-544 expression was significantly down-regulated in sphere-forming HCC cells during enrichment culture of liver cancer stem cells (P<0.05), and overexpression of miR-544 inhibited the sphere-formation of HCC cells (P<0.05). Conclusion MiR-544 expression is reduced in HCC tissues and can inhibit the malignant biological behaviors of HCC cells, indicating its inhibitory roles in the development and progression of HCC.