Genetic abnormalities in histone methyltransferases (HMTs) frequently occur in diffuse large B-cell lymphoma (DLBCL) and are related to its progression. SET and MYND domain containing 3 (SMYD3) is an HMT that is upregulated in various tumors and promotes their malignancy. However, to the best of our knowledge, the function of SMYD3 in DLBCL has not been investigated thus far. In the present study, 22 HMT genes related to cancer development were first selected according to current literature, and it was found that high SMYD3 expression was significantly associated with poor progression-free survival in patients with DLBCL. SMYD3 protein levels were upregulated and positively associated with poor prognosis and poor responsiveness to chemotherapy in patients with DLBCL. Functional examinations demonstrated that SMYD3 increased cell proliferation and the flux of aerobic glycolysis in DLBCL cells in vitro and in vivo and decreased cell sensitivity to doxorubicin in vitro. Moreover, SMYD3 could directly bind to specific sequences of Pyruvate Kinase M2 (PKM2) and promote DLBCL cell proliferation and aerobic glycolysis via H3K4me3-mediated PKM2 transcription. Clinically, SMYD3 expression positively correlated with that of PKM2, and high SMYD3 was significantly associated with high maximum standardized uptake value (SUVmax) detected by [(18)F]-fluorodeoxyglucose ((18)F-FDG) PET/computed tomography (PET/CT) in DLBCL samples. Concomitant expression of SMYD3 and PKM2 positively correlated with poor progression-free and overall survival in patients with DLBCL and may serve as novel biomarkers in DLBCL.