Meat tenderness has long been cited as one of the most important attributes of meat which greatly influences consumer acceptability. Different methods are available for tenderizing process, although they have not fulfilled meat processor requirements in terms of production costs and modification of the meat properties. So, the present study was designed to investigate the effects of ultrasonic radiation, alone and coupled with papain on the tenderness and other physicochemical and functional properties of Longissimus dorsi muscle. For this purpose, the effects of ultrasound, both singly and in combination with papain treatment, were investigated on pH, proteolytic activity, filtering residues, Warner–Bratzler shear force, meat textural profile (TPA), water-holding capacity, cooking loss, emulsion capacity and stability and gelling property. In addition, to evaluate the effect of ultrasound on enzyme penetration, enzyme activity, micro-structural changes and SDS-PAGE test were conducted on samples. Samples were treated by ultrasound using a probe at 100 and 300 W for 10, 20, and 30 min in the presence and absence of 1% papain solution. The results indicated that the effect of ultrasonic radiation on pH depends on time and radiation power. Enzyme and ultrasound treatment at 100 W did not have significant effect on pH but when they applied together, pH changed significantly (p 0.05). Application of enzyme, either singly or coupled with ultrasound radiation significantly (p 0.05) decreased the filtering residue, cooking loss and gelling properties. Ultrasound and enzyme treatments and their combination significantly increased the enzyme activity, water holding capacity, emulsion capacity and stability (p 0.05). While enzyme treatment was able to bring about a significant enhancement on these properties, combined treatment gave a rise to a greater enhancement in the enzyme activity, water holding capacity, emulsion capacity and stability. It was also found that ultrasound was able to reduce shear force by about 17%. Even, better results were obtained when it was combined with enzyme, as meat tenderness was improved by about 47%. Another finding was the severe microstructural damage. The ultrasonically induced cavitation and protease activity of papain were observed to cause cell wall disruption accompanied by the release of intracellular constituents that results in significant changes in the muscle microstructure. Another interesting result was the effect of ultrasonic radiation on improving mass transfer and enhancing enzyme penetration in the deeper layers of meat. So, ultrasonic radiation can be used toachieve the desired penetration depth of enzyme in a shorter time compared to other methods. Survey of extracted proteins by SDS-PAGE electrophoresis also showed that smaller protein bands were created. Finally, among the treatments explored in this study, the best results was obtained when the combined treatment was applied at ultrasonic power of 100 W for 20 min. It may thus be concluded that the combination of ultrasound radiation and papain treatment could be used as a promising technique for improving beef physicochemical and functional properties. Keywords: Tenderness, Papain, Ultrasound, Longissimus dorsi.