The protein digestibility of beef at three prefreezing temperatures (freezing at -20 °C, F20; freezing at -50 °C, F50; and freezing at -70 °C, F70) and aging periods (4, 14, and 28 days) was investigated using an in vitro infant digestion model. The increased cathepsin B activity in the frozen-then-aged treatments (P < 0.05) resulted in a higher content of 10% trichloroacetic acid-soluble α-amino groups than in the aged-only group on days 14 and 28 (P < 0.05). F50 had the most α-amino groups in the digesta and digested proteins under 3 kDa on day 28 (P < 0.05), with the disappearance of actin band in the digesta electrophoretogram. The secondary and tertiary structures of myofibrillar proteins revealed that F50 underwent irreversible denaturation (P < 0.05), especially in the myosin fraction, while F20 and F70 showed protein renaturation during aging (P < 0.05). In general, prefreezing at -50 °C then aging can improve the in vitro protein digestibility of beef through freezing-induced structural changes.