Aspergillus flavus is a common fungal pathogen of plants, animals and humans. Recently, many genes of A. flavus have been reported involving in regulation of pathogenesis in crops, but whether these genes are involved in animal virulence is still unknown. Here, we used a previous easy-to-use infection model for A. flavus based on mouse model by intravenous inoculation of A. flavus conidia. The outcome of infections in mice model showed that A. flavus NRRL3357 and laboratory strain CA14 PTS were both in dose dependent manner and highly reproducible. The progress of disease could be monitored by mice survival and histology analysis. Fungal burden analysis indicated it was gradually decreased within 7 days after infection. Moreover, aspergillosis caused by A. flavus significantly up-regulated gene expression levels of immune response mediators, including INF-γ, TNF-α, Dectin-1 and TLR2. Furthermore, the defined deletion A. flavus strains that previously displayed virulence in crop infection were also determined in this mouse model, and the results showed comparable degrees of infection in mice. Our results suggested that intravenous inoculation of conidia could be a suitable model for testing different A. flavus mutants in animal virulence. We hope to use this model to determine distinct A. flavus strains virulence in animals and study novel therapeutic methods to help control fungus diseases in the future.