Dengue virus (DENV) infection and replication induces oxidative stress, which further contributes to the progression and pathogenesis of the DENV infection. Modulation of host antioxidant molecules may be a useful strategy for interfering with DENV replication. In this study, we showed that induction or exogenous overexpression of heme oxygenase-1 (HO-1), an antioxidant enzyme, effectively inhibited DENV replication in DENV-infected Huh-7 cells. This antiviral effect of HO-1 was attenuated by its inhibitor tin protoporphyrin (SnPP), suggesting that HO-1 was an important cellular factor against DENV replication. Biliverdin but not carbon monoxide and ferrous ions, which are products of the HO-1 on heme, mediated the HO-1-induced anti-DENV effect by non-competitively inhibiting DENV protease, with an inhibition constant (Ki) of 8.55 ± 0.38 μM. Moreover, HO-1 induction or its exogenous overexpression, rescued DENV-suppressed antiviral interferon response. Moreover, we showed that HO-1 induction by cobalt protoporphyrin (CoPP) and andrographolide, a natural product, as evidenced by a significant delay in the onset of disease and mortality, and virus load in the infected mice's brains. These findings clearly revealed that a drug or therapy that induced the HO-1 signal pathway was a promising strategy for treating DENV infection.