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Emerging evidence shows that HO-1 also exhibits antiviral activity against severe acute respiratory syndrome coronavirus 2, human immunodeficiency virus, hepatitis B virus, and Ebola virus. Its antiviral effects are mediated not only by its enzymatic function but also through the modulation of interferon-related pathways, thereby inhibiting viral replication. In this study, we investigated the antiviral effects of HO-1 on canine coronavirus (CCoV) and canine influenza virus (CIV) H3N2 using cell-based assays. To determine whether HO-1 suppresses CCoV and CIV, cells were treated with hemin to induce HO-1 expression. Hemin treatment successfully induced HO-1 expression in A72 and Madin-Darby canine kidney cells, resulting in the suppression of CCoV and CIV replication. The canine HO-1 gene was cloned into an expression vector and transfected into cells to achieve transient overexpression. Recombinant canine HO-1 protein was expressed in Escherichia coli and purified using an expression vector. HO-1 overexpression suppressed CCoV and CIV replication in cells. Following viral infection, treatment with purified HO-1 protein led to a reduction in viral protein levels. Therefore, both HO-1 expression and exogenous protein treatment effectively inhibited CCoV and CIV replication. Elevated HO-1 protein levels consistently reduced viral RNA and protein expression in vitro. 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