Background In our previous study, a venom-based peptide named Gonearrestide (also named P13) was identified and demonstrated with an effective inhibition in the proliferation of colon cancer cells. In this study, we explored if P13 and its potent mutant M6 could promote the proliferation of human embryonic stem cells and even maintain their self-renewal. Methods The structure-function relationship analysis on P13 and its potent mutant M6 were explored from the molecular mechanism of corresponding receptor activation by a series of inhibitor assay plus molecular and dynamics simulation studies. Results An interesting phenomenon is that P13 (and its potent mutant M6), an 18AA short peptide, can activate both FGF and TGFβ signaling pathways. We demonstrated that the underlying molecular mechanisms of P13 and M6 could cooperate with proteoglycans to complete the “dimerization” of FGFR and TGFβ receptors. Conclusions Taken together, this study is the first research finding on a venom-based peptide that works on the FGF and TGF-β signaling pathways to maintain the self-renewal of hESCs.