Aspergillus fumigatus is a fungal pathogen of major clinical importance. However, little is known about the role of human bronchial epithelial cells (HBECs) during A. fumigatus conidia induced inflammation. Here, we show that differentiated respiratory epithelial cells recognise inactivated resting conidia but not swollen conidia or hyphae, resulting in the induction of the interferon (IFN)-β signalling pathway and the expression of IFN-β-inducible genes, such as IFN-γ-inducible protein (IP)-10. This induction was internalisation dependent. We identified double-stranded conidial RNA recognised by Toll-like receptor-3 as a factor responsible for the expression of IFN-β and IP-10. Inhibition of receptor-interacting protein-1/TANK-binding kinase-1, known to mediate IFN-β signalling, was sufficient to inhibit the induction of IFN-β and IP-10 expression by conidia. Even though conidia induced the activation of nuclear factor (NF)-κB in HBECs, IP-10 expression was only partially dependent on NF-κB signalling. These results provide evidence that respiratory cells are activated by the double-stranded RNA of resting conidia and initiate a first immune response to inhaled conidia in an IFN-β-dependent manner.