Spider venom is one of valuable resources for the development of novel anticancer drugs. In this study, we focused on a novel linear amphipathic α-helical anticancer peptide LVTX-9, which was derived from the cDNA library of the spider Lycosa vittata venom gland. The cytotoxicity of LVTX-9 against cancer cells was significantly lower than those of most anticancer peptides reported. It’s IC50 was determined to ～90 μM). Fatty acids modification is a promising strategy to improve peptide performance. Therefore, in order to enhance the cytotoxic activity of LVTX-9, fatty acids modification of this peptide was performed, and five different carbon chain length lipopeptides named LVTX-9-C12-C20 were produced. Among them, the lipopeptide LVTX-9-C18 showed the highest cytotoxic activity to B16-F10 cells whether in serum or serum-free medium. Most importantly, the cytotoxic activity of LVTX-9-C18 was improved ～20 times compared to that of LVTX-9. Subsequently, assays including SEM, trypan blue staining, LDH leakage assay, and hemolytic activity indicated the direct cell membrane disruption was the main mechanism of LVTX-9-C18 to induce cancer cell death. Furthermore, the LVTX-9-C18 also showed strong cytotoxicity to 3D B16-F10 spheroids, which indicated it might be a promising lead for developing anticancer drugs.