Rapeseed peptides were prepared by means of the combined methods of the laboratory bacteria enzyme synergy and the solid-state fermentation of rapeseed meal. The rapeseed peptides were separated and purified with the tumor cell in vitro anti-proliferative activity as an index. Moreover, a kind of rapeseed peptide component RSP-4-3-3 (rapeseed anti-tumor peptide RSP-4-3-3) with high activity was selected. Furthermore, by using reversed-phase high performance liquid chromatography (RP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS/MS), the analysis result of its possible amino acid sequence showed that it was Trp-Thr-Pro (408.2 Da). Inverted microscope observation technology and western blot experiments were applied to explore the antitumor impact of the rapeseed peptide RSP-4-3-3 on tumor cells. The results showed that the rapeseed antitumor peptide RSP-4-3-3 could significantly change the morphological features of the HepG2 cells in vitro and cause apoptosis, thus inhibiting the proliferation of the HepG2 cells.