To analyze the inhibitory effect of dihydrotanshinone I (DiⅠ) on porcine epidemic diarrhea virus (PEDV) in vitro, the cytotoxicity of DiⅠ on Vero and IPEC-J2 cells was first determined by CCK-8 assay, and the results showed no significant toxicity at the concentrations used in subsequent experiments. Subsequently, multiple methods, including quantitative RT-PCR, Western blot, indirect immunofluorescence, and virus titer assay, demonstrated that DiⅠ significantly inhibited PEDV gene expression and viral protein synthesis in a dose-dependent manner. Mechanistic studies revealed that DiⅠ effectively interfered with key stages of the PEDV life cycle, including viral entry, replication, and release. Comparison of different administration modes indicated that, except for pre-treatment, full-treatment, co-treatment, and post-treatment all effectively suppressed viral replication, suggesting the therapeutic potential of DiⅠ even after infection. Molecular docking method was used to evaluate the interaction between DiⅠ and PEDV Mpro. In conclusion, DiⅠ exhibits potent anti-PEDV activity and represents a promising candidate drug against PEDV.