S100A9 is differentially expressed in various cell types and is associated with the development, progression and metastasis of various cancers. However, the expression, distribution, and clinical significance of S100A9 in hepatocellular carcinoma (HCC) remain unclear. In the present study, The Cancer Genome Atlas (TCGA) database was used to examine S100A9 gene expression in HCC; we found that S100A9 expression was associated with HCC prognosis. In addition, S100A9 protein expression was assessed by immunohistochemistry analysis of tissues from 382 HCC patients. We found that the infiltration of S100A9+ cells in both tumor and nontumor tissues could predict poor overall survival (P = 0.0329, tumor; P = 0.0003, nontumor) and a high recurrence risk (P = 0.0387, tumor; P = 0.0015, nontumor) in our tissue microarray analysis. Furthermore, immunofluorescence double staining revealed that the primary S100A9-expressing cells in adjacent nontumoral tissue were CD15+ neutrophils, and both CD68+ macrophages and CD15+ neutrophils expressed S100A9 in HCC tumor tissues. Taken together, the results suggest that high S100A9+ cell density predicts a poor prognosis in HCC patients, and S100A9 expression could potentially serve as an independent prognostic marker for HCC.