Odorant-binding proteins (OBPs) are abundant soluble proteins secreted in the nasal mucus of a variety of species which are believed to be involved in the transport of odorants towards olfactory receptors. In this study, we report the functional characterization of mouse OBP5 (mOBP5). mOBP5 was recombinantly expressed as a hexahistidine-tagged protein in bacteria and purified by metal affinity. Oligomeric state and secondary structure composition of mOBP5 was investigated using gel filtration and circular dichroism spectroscopy. Fluorescent experiments revealed that mOBP5 interacts with the fluorescent probe N-phenyl naphthylamine (NPN) with a micromolar affinity. Competitive binding experiments with 40 odorants indicated that mOBP5 binds a restricted number of odorants with a good affinity. Isothermal titration calorimetry (ITC) confirmed that mOBP5 binds these compounds with association constants in the micromolar range. Finally, protein homology modelling and molecular docking analysis revealed the amino acid residues of mOBP5 guiding its binding properties.