Coronaviruses have undergone evolutionary changes and mutations in response to the immune pressures exerted by vaccines and environmental factors, resulting in more severe consequences during breakthrough infections. Nevertheless, the specific correlation between the evolutionary mutations of coronaviruses and immune pressures remains ambiguous. Swine coronavirus-porcine epidemic diarrhea virus (PEDV)-has existed for decades. This study utilized preparation of polyclonal antibodies against the PEDV and identified critical neutralizing epitopes through serial passaging. Then, the recombinant mutated strains were successfully constructed. experiments confirmed the ability of the rA1273P strain to escape neutralization by polyclonal antibodies. Both cell studies and animal experiments revealed that the strain maintains virulence and pathogenicity while evading antibody pressure post-vaccination. The pathogenicity of the strain while evading immune pressure is comparable to wild-type strains. A comparison of the S protein gene between vaccine strains and clinical strains identified mutations in 1273 amino acid positions in clinical strains. In conclusion, this study identified a novel PEDV S protein neutralizing site under immune pressure through serial passaging, indicating that the 1,273th amino acid position is prone to mutation under prolonged antibody pressure, enhancing the virus's ability to escape hosts. This study offers new insights into the interpretation of coronavirus escape immune pressure and provides technical support for monitoring and predicting the variation and evolution of coronavirus.IMPORTANCECoronaviruses represent an ongoing public health threat because of high variability. Since 2010, the emergence of highly pathogenic porcine epidemic diarrhea virus (PEDV) strains has resulted in significant economic losses to the global pig industry. PEDV undergoes evolution and mutation under external immune pressure, rendering it an increasingly challenging target for prevention and control measures. Here, we prepared the polyclonal antibodies against PEDV and identified a novel neutralization epitope on the S protein (1,273th amino acids) through serial passaging. Furthermore, our findings indicate that the mutation of A1273P in the S protein did not alter the virulence of the PEDV but significantly enhanced its ability to escape and infect the host and . Finally, we found that the 1,273 amino acid position of the S gene has been mutated to varying degrees in clinical PEDV strains. This work provides a specific correlation between the evolutionary mutations of coronaviruses and immune pressures.