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Simple Summary: Odorant-binding proteins are soluble proteins abundantly secreted in the nasal mucus of vertebrates. Although their physiological functions are not known, these proteins are suspected to have a carrier role in solubilizing and carrying hydrophobic odorant molecules to the olfactory receptors. Here, we describe the expression of functional mouse mOBP5. The protein produced using bacteria was purified and characterized. We investigated its binding properties using a fluorescent competitive assay and microcalorimetry. Molecular docking experiments revealed hydrophobic residues in the binding cavity potentially involved in the stabilization of the odorant, thus explaining its binding properties. Odorant-binding proteins (OBPs) are abundant soluble proteins secreted in the nasal mucus of a variety of species that are believed to be involved in the transport of odorants toward 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 using metal affinity chromatography. The oligomeric state and secondary structure composition of mOBP5 were investigated using gel filtration and circular dichroism spectroscopy. Fluorescent experiments revealed that mOBP5 interacts with the fluorescent probe N-phenyl naphthylamine (NPN) with micromolar affinity. Competitive binding experiments with 40 odorants indicated that mOBP5 binds a restricted number of odorants with good affinity. Isothermal titration calorimetry (ITC) confirmed that mOBP5 binds these compounds with association constants in the low micromolar range. Finally, protein homology modeling and molecular docking analysis indicated the amino acid residues of mOBP5 that determine its binding properties. [ABSTRACT FROM AUTHOR]