Background: Colorectal Cancer (CRC) is one of the leading causes of death worldwide. MicroRNA-34a (miR-34a), a tumor suppressor gene, is known to be down-regulated in CRC cell lines and recently shown to be a cell-fate determinant in early-stage dividing colon cancer stem cells. However, its prognostic significance is unclear. Methods: MiR-34a detection was performed by in situ hybridization on a CRC tissue microarray (n=127; stage I, 21 patients; II: 42; III: 33; IV: 31). Its expression was graded as negative (no signal), low (expression in 1-19% of cancer cells), moderate (20-49% of cancer cells), and strong (50-100% of cancer cells). The correlations between miR-34a expression and EGFR, osteopontin (OPN), p53, Ki67, LEF1, VEGF, COX2, MMR, stage and grade were evaluated by chi-squared test and Spearman's rank correlation coefficient. Kaplan-Meier survival analysis, log-rank test and Cox regression model were used to assess the association of miR-34a expression with recurrence-free survival (RFS) and disease specific survival (DSS). Results: MiR-34a expression had moderate positive correlation with genes associated with tissue proliferation and invasion, including Ki67 (Spearman's r=0.28, p=0.002), and weak correlation with EGFR (r=0.2, p=0.02) and LEF1 (r=0.18, p=0.039). In the subgroup of patients (n=75; stage II/III) with negative OPN expression (n=25), weak or negative miR-34a expression was associated with worse RFS (HR=4.1; 95%CI=1.1-15.9; p=0.036) and DSS (HR=10.5; 95%CI=1.2-94.5; p=0.036). Conclusions: Our results suggest that down-regulated or absent expression of miR-34a correlates with worse RFS and DSS in stage II - III CRC patients with negative OPN expression. Further investigation of miR34a in prospective randomized studies is warranted to establish its role as a prognostic factor for CRC outcome.