Cathepsin D plays a potential role in the degradation of muscle proteins, thereby influencing their functional properties and structural integrity. The changes in functional properties and microstructure of muscle proteins in Parapenaeopsis hardwickii treated with cathepsin D were investigated through in vitro simulation. Myofibrillar, actin, and sarcoplasmic proteins were extracted from P. hardwickii muscles and were divided into the experimental (treated with cathepsin D) and control (which received no enzyme treatment) groups. All samples were incubated at 50 ℃ for 30 min. The turbidity, surface hydrophobicity, endogenous fluorescence, free amino acid content, and particle size were measured. Compared with the control groups, the experimental groups exhibited significantly higher turbidity and surface hydrophobicity (P<0.05) but lower endogenous fluorescence, free amino acid content, and particle size. Turbidity values of the three proteins in the experimental groups were 0.45, 0.45, and 0.42, respectively. The amounts of bound bromophenol blue were 9.66, 56.30, and 2.34 μg, respectively, exceeding those noted in the control groups. Furthermore, the particle sizes of the three proteins in the experimental groups were 819.8, 3 230.3, and 1 459.8 nm smaller than those of the control groups. The levels of antioxidant amino acids (such as glycine and alanine) in the three protein types within the experimental groups were also significantly lower than those in the control group. These results indicate that cathepsin D effectively degrades myofibrillar, actin, and sarcoplasmic proteins in the muscles of P. hardwickii, thereby disrupting their functional properties and promoting muscle softening and autolysis. These findings provide a theoretical basis for in vitro simulation of cathepsin D and storage quality control of P. hardwickii.