CK-666, an inhibitor of the actin-related protein complex 2/3 (Arp2/3), can suppress lamellipodia formation and cell migration. However, research on its application in tumor therapy is still limited. Using RNA-seq, we clustered and analyzed the functions of differentially expressed mRNAs in CK-666-treated tumor cells. Interestingly, the differentially expressed genes related to CK-666 were closely associated with exosomes and autophagy. Through Western blot, we confirmed that CK-666 promoted the high expression of exosome and autophagy markers in tumor cells. Transmission electron microscopy results indicated the appearance of extracellular vesicles larger than exosomes. Scanning electron microscopy findings revealed that CK-666 inhibited the formation of intercellular tunneling nanotubes (TNTs). Fluorescent staining further revealed that CK-666 induced the formation and secretion of CD63-positive vesicles within the tunnels of retraction fibers (RFs). In vitro experiments verified that CK-666 preferentially inhibited fibroblasts in 3D tumorspheres. In the tumor 3D-Histoculture Drug Response Assay (3D-HDRA), it was found that CK-666 could suppress the activity of isolated tumor tissues. Moreover, our study discovered that the combination of CK-666 and docetaxel (DTX) significantly enhanced DTX sensitivity. In summary, our results suggest that CK-666 may play an oncogenic role by regulating autophagy, TNTs, and extracellular vesicles formation.