The motile characteristics and mechanisms that drive the dissemination of diffuse large B-cell lymphoma (DLBCL) are elusive. Here, we show that DLBCL initiates dissemination through activating STAT3-mediated amoeboid migration. Mechanistically, STAT3 activates RHOH transcription, which competes with the RhoGDP dissociation inhibitor RhoGDIγ to activate RhoA. In addition, activated STAT3 regulates microtubule dynamics and releases ARHGEF2 to activate RhoA. Both the JAK inhibitor ruxolitinib and the microtubule stabilizer Taxol suppress DLBCL cell dissemination in vivo. A clinical DLBCL sample analysis shows that STAT3-driven amoeboid movement is particularly important for the transition from stage I to stage II. This study elucidates the mechanism of DLBCL dissemination and progression and highlights the potential of combating advanced DLBCL with a JAK/STAT inhibitor or microtubule stabilizer to reduce DLBCL motility; these findings may have a great impact on the development of patient-tailored treatments for DLBCL.