Intracellular tau aggregates composed of neurofibrillary tangles (NFTs) are a defining feature of Alzheimer's disease (AD). Increased expression of heme oxygenase-1 (HO-1) is a common phenomenon in AD. Interestingly, the spatial distribution of HO-1 expression is essentially identical to that of pathological accumulation of tau in AD. In this study, we developed a new transgenic mouse overexpressing HO-1, called CAG-HO-1 Tg mice, to explore the relationship between HO-1 and tau aggregation. In this model, we found that long-term overexpression of HO-1 significantly promoted tau aggregation in brain, by analyzing changes in morphology and insoluble tau expression levels. Moreover, our research provides the first in vivo evidence that HO-1 can enhance iron loading and tau (Ser199/202/396) phosphorylation in brains of transgenic mice. Cellular evidence indicates that HO-1 can induce the phosphorylation of tau through iron accumulation in Neuro2a cells stably transfected with HO-1. Our data suggest that long-term overexpression of HO-1 can promote tau aggregation. This mechanism involves excessive iron production mediated by HO-1 overexpression, which induces tau phosphorylation. Our results provide a potential pathway for the pathogenesis of tauopathies, which remains largely unknown.