We analyzed the clinicopathologic implication of A20/tumor necrosis factor α-induced protein 3 deletion in diffuse large B-cell lymphoma (DLBCL) using fluorescence in situ hybridization, according to germinal center B-cell (GCB) versus non-GCB/activated B-cell (ABC) phenotypes and rituximab treatment. Excluding primary central nervous system (CNS) and Epstein-Barr virus (EBV)-positive lymphomas, 134 DLBCLs were analyzed. A20 was deleted in 23.1% (31/134) of DLBCLs including 21.6% (29/ 134) of monoallelic and 1.5% (2/134) of biallelic deletion, with no predilection for GCB versus non-GCB/ABC. In univariate analysis, A20 deletion was marginally associated with favorable prognosis in the rituximab-treated subgroup (n = 109; p = 0.0454), non-gastrointestinal lymphoma (n = 108; p = 0.0320) and nodal lymphoma (n = 46; p = 0.0411). In multivariate analysis in rituximab-treated DLBCL, MUM1 and international prognostic index (IPI) were independent prognostic factors (p = 0.021 [IPI]; p = 009 [MUM1]) with a marginally favorable prognostic effect for A20 deletion (p = 0.047). Taken together, A20 deletion was observed in similar frequencies in GCB and non-GCB/ABC, and was not a poor prognostic factor in DLBCL.