Hybrid proline-rich proteins (HyPRPs) are cell wall-localized proteins, and are frequently responsive to environmental stresses. The coding sequence of a HyPRP cDNA was isolated from Medicago falcata, a forage crop that shows cold and drought tolerance. The predicted MfHyPRP contains a proline-rich domain at N-terminus after the signal peptide and a conserved eight-cysteine motif at the C-terminus. Higher level of MfHyPRP transcript was observed in leaves than in stems and roots under control conditions, while more MfHyPRP transcript was induced in leaves and stems than in roots after cold treatment. Levels of MfHyPRP transcript and MfHyPRP protein in leaves were induced by cold, dehydration, abscisic acid (ABA), hydrogen peroxide (H2O2) and nitric oxide (NO), but not responsive to salt stress. The cold- or dehydration-induced expression of MfHyPRP was blocked by scavenger of NO, but not affected by inhibitor of ABA biosynthesis or scavenger of H2O2. The results indicated that NO, but not ABA and H2O2, was essential in the cold- and dehydration-induced expression of MfHyPRP. Overexpression of MfHyPRP in tobacco led to increased tolerance to freezing, chilling and osmotic stress as well as methyl viologen-induced oxidative stress. The increased cold and osmotic stress tolerance was proposed to be associated with improved protection against oxidative damages. It is suggested that NO mediates cold- and dehydration-induced expression of MfHyPRP that confers tolerance to abiotic stress.