We hypothesised that comparing the protein mixture in bronchoalveolar lavage fluid (BALF) between humans and mice may lead to mechanistic insights into common and divergent pathways that evolved in each species. BALF from four humans and six mice was pooled separately and underwent identical shotgun proteomic analysis. Functional and network analysis was applied to identify overlapping and distinct pathways enriched in the BALF. Follow-up experiments using Western analysis in unpooled BALF samples were performed. We identified 91 unique proteins in human and 117 unique proteins in mouse BALF samples. Functional analysis of the proteins revealed conservation of several key processes between the species, including defence response. Oxidative stress response, however, was selectively enriched only in mouse BALF. Differences in the expression of peroxiredoxin-1, a key member of the defence pathway against oxidative injury, were confirmed between normal human and mouse BALF and in models of lung injury. A computational proteomics approach of mouse and human BALF confirms the conservation of immune and defence-mediated pathways while highlighting differences in response to oxidative stress. These observations suggest that the use of mice models to study human lung disorders should be undertaken with an appreciation of interspecies variability.