〔Abstract〕 Objective Previous study has found that peptide301LARSLKT307can significantly inhibit fetal cardiac developmentin vivo. This study intends to further explore the role and mechanism of301LARSLKT307in inhibiting myocardial differentiation at P19 cell. Methods The structure, conservation, physical and chemical property and precursor proteins of peptide301LARSLKT307were analyzed by PepDraw Tool, Uniprot and ProtParam online. The entry of FITC-labeled peptide301LARSLKT307into P19 cells was observed under a fluorescence microscope. After adding peptide301LARSLKT307, the differentiation of P19 cells was observed by inverted microscope. The expression of troponin cTnT and transcription factor GATA4 were detected by qPCR and Western blot assay to analyze the specific marker gene of myocardium. The expressions of Notch1, Hey1 and Hey2 were detected by qPCR to analyze the effect of peptides on Notch1 pathway in P19 cell. Results Bioinformatics analysis showed that the peptide was located at the 301-307 amino acid site of its precursor protein Talin, which is a small molecule peptide with stable structure, high lipophilicity and high sequence conservation. FITC-labeled peptide301LARSLKT307can enter the P19 cell under the fluorescence microscope. Compared with the control group, the cells in the peptide group were partially necrotic, with disordered growth and morphology. The qPCR and Western blot assay showed that the differentiation related genes GATA4 and cTnT in the peptide group both decreased at different levels. The qPCR showed that the Notch-related genes Notch1, Hey1 and Hey2 in the peptide group decreased at different levels. Conclusion Peptide301LARSLKT307may inhibit the biological function of myocardial differentiation by inhibiting the expression of key factors of Notch1 pathway.