Introduction: This study was designed to investigate the roles of STAT3 and SMYD3 in chronic lymphocytic leukemia and the regulatory relationship between STAT3 and SMYD3 in chronic lymphocytic leukemia. Material and methods: The expression of STAT3 and SMYD3 was determined by RT-qPCR and western blot in chronic lymphocytic leukemia samples and cells (MEC1, CLL). Small interfering RNA was used to knock down the mRNA level of STAT3 and the pcDNA3.1-SMYD3 plasmid was used to construct a SMYD3 overexpression model. An MTT assay was performed to evaluate cell proliferation. A transwell assay was used to detect cell invasion ability. Afterwards, a luciferase reporter assay and chromatin immunoprecipitation experiment (ChIP assay) were applied to confirm the correlation between STAT3 and SMYD3. Results: STAT3 was highly expressed in chronic lymphocytic leukemia mononuclear cells and cancerous cell lines. STAT3 knockdown dramatically inhibited the mRNA and protein expression of SMYD3 in MEC1 and CLL cell lines. The luciferase reporter assay combined with the ChIP assay revealed that STAT3 bound to the promoter region of STAT3 and contributed to the transcription of SMYD3. Knockdown of STAT3 positively correlated with inhibition of cell proliferation and invasion abilities, while overexpression of SMYD3 negatively correlated with inhibition of cell proliferation and invasion. Conclusions: STAT3 may promote chronic lymphocytic leukemia progression through elevating SMYD3 expression. Targeting STAT3 and SMYD3 may be a potential therapeutic strategy for chronic lymphocytic leukemia.