This study investigated the effects of NO-enhanced Pediococcus pentosaceus YT2 on the tenderness of post-mortem beef. Increasing the concentration of YT2 increased the myofibrillar fragmentation index (MFI) and decreased the shear force of beef. Fourier transform infrared spectroscopy revealed that YT2 caused changes in protein-water interactions. As the concentration of YT2 was increased, the content of β-sheet increased from 33.12 to 39.85%, of β-turn increased from 13.39 to 14.33%, and of α-helix decreased from 53.49 to 46.37%. Low-field nuclear magnetic resonance showed that incorporating YT2 at 6-11 log CFU/g could effectively influence the moisture distribution status and improve the water holding capacity of beef. SDS-PAGE revealed that myofibrillar proteins were degraded to varying degrees in all groups. Furthermore, Western blotting demonstrated that desmin and troponin-T were significantly degraded (P < 0.05) in YT2-treated samples at 168 h post-mortem aging, and the degree of degradation was affected by YT2 concentration. The results closely aligned with those of the MFI and shear force. Overall, the data indicated that YT2 participated in the degradation of key myofibrillar proteins to improve beef tenderization.