The PI3K/AKT pathway might be involved in the development of some certain diffuse large B-cell lymphoma (DLBCL) by as-yet unclear mechanisms. PIK3CA mutations in exons 9 and 20 were investigated in 76 primary human DLBCLs, 3 DLBCL cell lines (LY1, LY8, and LY10), and 9 related samples using polymerase chain reaction-based sequence analysis to assess the possible relevance of PIK3CA mutations in DLBCL to the PI3K/AKT pathway activation. AKT phosphorylation (pAKT) of 3 DLBCL cell lines and 76 primary DLBCL samples was also detected by Western blot and immunohistochemistry. All 3 cell lines showed high levels of pAKT, and 72.4% (55/76) of the DLBCLs expressed pAKT at various levels, indicating the activation of AKT. However, no mutation was found in exons 9 or 20 in PIK3CA in any of the 3 cell lines. Only 1 out of 76 primary DLBCLs (1.32%) harbored an exon 9 mutation, and no exon 20 mutation was detected. The case with mutations contained 3 mutation points. One was c.1634A>C resulting in E545A, which was in a previously reported hotspot. The other 2 were novel c.1658G>C and c.1659delT frameshift mutations. We conclude that the PI3K/AKT pathway is activated in DLBCL and that PIK3CA is rarely mutated in DLBCL, indicating there could be some other PI3K-pathway activation mechanisms operative in DLBCL.