This study was aimed to evaluate the proliferation-associated antigen Ki-67 expression in diffuse large B cell lymphoma (DLBCL) and its clinical significance. The Ki-67 expression and its correlation with prognosis in 50 patients with DLBCL treated with rituximab plus CHOP (R-CHOP) between January 2008 and December 2010 were analyzed retrospectively. The results indicated that there was no significant relationship between Ki-67 expression and clinical features, including age, sex, staging, B symptoms, LDH level, IPI, extranodal site involvement, presence of bulky tumors (>10 cm in diameter), bone marrow involvement, GCG nor GCB type, or response to first line treatment. The median survival was 50 months and 15 months in low Ki-67 expression group (<85%) and in high Ki-67 expression group ( ≥ 85%) respectively. The overall survival (OS) and progress-free survival (PFS) in low Ki-67 expression group were obviously longer than that in high Ki-67 expression group (P = 0.001; P = 0.027). In univariate analysis, the clinical factors associated with OS included Ann Arbor staging and Ki-67 expression. The clinical factors associated with PFS included Ann Arbor staging. IPI and Ki-67 expression. In multivariate analysis. The Ki-67 expression level was an independent prognostic factor for OS (HR = 4.90; 95% CI, 1.456-16.511; P = 0.0103). It is concluded that Ki-67 expression level seems to be an effective marker for evaluation of DLBCL prognosis.