A natural chalcone, cardamonin (2′,4′-dihydroxy-6′-methoxychalcone; CDN) was isolated from the seeds of Alpinia katsumadai Hayata, which has been traditionally used to treat stomach aches. CDN has been reported to possess various pharmacological properties, including anticancer and anti-inflammatory effects. This study evaluated the antiviral activity of CDN against human coronavirus HCoV-OC43 and determined the mode of action in HCoV-OC43-infected human lung cell lines (MRC-5 and A549 cells). CDN significantly inhibited HCoV-OC43-induced cytopathic effects with an IC50 of 3.62 μM and a CC50 of >50 μM, resulting in a selectivity index of >13.81. CDN treatment reduced the level of viral RNA and the expression of spike and nucleocapsid proteins in HCoV-OC43-infected cells as determine through qRT-PCR and Western blot analysis. Additionally, the activation of p38 mitogen-activated protein kinase (MAPK) by anisomycin decreased viral protein expression, whereas an inhibitor of p38 MAPK signaling, SB202190, increased viral protein expression. CDN also amplified and extended the p38 MAPK signaling pathway in HCoV-OC43-infected cells. In conclusion, CDN inhibited HCoV-OC43 infection by activating the p38 MAPK signaling pathway and has potential as a therapeutic agent against human coronavirus.