MicroRNA-21 (miR-21) has been ascribed a key role in many cellular processes, e.g. tumorigenesis via inhibition of target gene expression. However, its role in diffuse large B-cell lymphoma (DLBCL) is still unclear, and there are no in-depth studies on the relationship between miR-21 and the cellular phenotype of DLBCL. In this study, we investigated the expression and role of miR-21 in the regulation of cell biological processes in DLBCL. Firstly, miR-21 expression was evaluated in three DLBCL cell lines by real-time quantitative reverse-transcription (qRT) polymerase chain reaction (PCR). Then, to determine the possible role of miR-21 in the biological and behavioral characteristics of DLBCL, we performed miR-21 knockdown by transfection with anti-miR-21. In addition, PDCD4 and PTEN were assessed by luciferase reporter assay, qRT-PCR, and Western blot. Our study revealed that miR-21 was significantly upregulated in activated B-cell-like DLBCL cells compared to germinal center-like DLBCL cells. We demonstrated that inhibition of miR-21 induced suppression of proliferation and invasion, as well as increased apoptosis in DLBCL. Moreover, knockdown of miR-21 increased PDCD4 and PTEN expression at the protein level but not at the mRNA level. In conclusion, miR-21 can regulate proliferation, invasion, and apoptosis, and thus it has a potential therapeutic application in DLBCL.