Objectives: Rituximab has significantly improved the survival of patients with DLBCL, especially those with non-germinal center B-cell-like (non-GCB) subtype. The impact of Ki-67 expression, an index of proliferation, on the clinical outcomes of patients with DLBCL has largely been unexplored. This study aimed to investigate whether Ki-67 expression is an indicator of outcome in DLBCL patients (especially non-GCB DLBCL patients) treated with standard chemotherapy combined with rituximab. Methods: Expression of Ki-67 protein was examined immunohistochemically in 118 tumor specimens from patients newly diagnosed with DLBCL and treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). Results: Overall survival (OS) and progression-free survival (PFS) were lower in patients with high Ki-67 expression than in those with low Ki-67 expression (3-year OS: 65.2% vs. 81.7%, P = 0.030; 3-year PFS: 56.4% vs. 73.3%, P = 0.020), similar in patients with GCB subtype and those with the non-GCB subtype (OS: P = 0.330; PFS: P = 0.287). According to Ki-67 expression status by immunophenotype subgroups, patients with high Ki-67 expression in non-GCB subgroup had the most unfavorable PFS and OS, comparing with the other three subgroups (P = 0.004 and P = 0.002, respectively). In multivariate analysis, non-GCB with high Ki-67 expression was an independent prognostic predictor of inferior survival in DLBCL patients treated with R-CHOP. Conclusion: For DLBCL patients with non-GCB DLBCL and high Ki-67 expression, the survival benefit from R-CHOP therapy is limited.