Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma and prognostic information is essential in finding the right treatment. This study evaluated the prognostic significance of Ki-67 in patients with DLBCL. Patients with DLBCL, treated with first-line R-CHOP, were retrospectively analyzed in groups of high (>70%) and low (≤70%) Ki-67. Parameters of interest were the international prognostic index (IPI), treatment response, progression-free survival (PFS) and overall survival (OS). A chi-squared test or Fisher's exact test was conducted to analyze categorical variables. Kaplan-Meier and log-rank tests were applied for survival analyses. Finally, a multivariate linear regression analysis was performed, including gender, Ki-67 ≤ 70% or >70%, IPI and presence of B symptoms. Overall, 58 patients were included. No significant association was found between Ki-67 status and IPI (p = 0.148) or treatment response (p = 0.373). Survival in patients with high Ki-67 was significantly inferior with respect to OS (p = 0.047) but not PFS (p = 0.138). Multivariate linear regression, however, yielded only IPI as a risk factor for OS. Future studies with larger patient cohorts are needed in order to elucidate the prognostic role of Ki-67 in patients with DLBCL treated with R-CHOP.