ObjectiveTo investigate the expression and clinical significance of serum exosomal microRNA(miRNA)-221-3p in hepatocellular carcinoma (HCC). MethodsA total of 66 patients who underwent surgery in Nanjing Drum Tower Hospital, Nanjing University Medical School, from February 2010 to December 2014 and were diagnosed with HCC were enrolled as HCC group, and 25 individuals who underwent physical examination in our hospital during the same period of time were enrolled as control group. The exosome extraction kit was used to isolate the exosomes in serum; Western blotting was used to measure the expression of exosome markers; a transmission electron microscope was used to observe the morphology of serum exosomes; quantitative real-time PCR was used to measure the expression of serum exosomal miRNA-221-3p. The Mann-Whitney U test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups. The Kaplan-Meier method and the log-rank test were used to analyze the association of the expression of serum exosomal miRNA-221-3p with prognosis, and the Cox proportional hazards regression model was used to analyze the influencing factors for prognosis. ResultsSerum exosomes were small membranous vesicles with a diameter of about 40-100 nm, and HSP70, Alix, and CD63 were expressed in the exosomes. The HCC group had significantly higher expression of serum exosomal miRNA-221-3p than the control group (U=354.00, P＜0.001). In the patients with HCC, the high expression of serum exosomal miRNA-221-3p was associated with tumor size (χ2=6.016, P=0.014), capsular invasion (χ2=7.580, P=0.006), and TNM stage (χ2=6.340, P=0012). In addition, the HCC patients with high expression of serum exosomal miRNA-221-3p had a significantly lower overall survival rate than those with low expression (χ2=17.105, P＜0.001). The multivariate analysis showed that the expression of serum exosomal miRNA-221-3p (hazard ratio ［HR］=2.434, 95% confidence interval ［CI］: 1.178-5.027, P=0.016) and tumor stage (HR=2.653, 95% CI: 1.222-5.760, P=0.014) were independent risk factors for the prognosis of patients with HCC. ConclusionThere is a significant increase in the expression of serum exosomal miRNA-221-3p in HCC patients, which provides a reference for the diagnosis and prognostic evaluation of HCC.