This study aimed to explore the effects of pre-slaughter transport stress on protein S-nitrosylation levels and S-nitrosylated proteome in postmortem pork longissimus thoracis (LT)muscle. Pigs (n = 16) were randomly divided into three-hour transport (high-stress, HS) and three-hour transport followed by three-hour resting treatments (low-stress control, LS). Results demonstrated that high transport stress levels induced nitric oxide (NO) overproduction by promoting NO synthase (NOS) activity and neuronal NOS (nNOS) expression, which thereby notably increased protein S-nitrosylation levels in postmortem muscle. Proteomic analysis indicated that 133 S-nitrosylation-modified cysteines belonging to 85 proteins were significantly differential, of which 101 cysteines of 63 proteins were higher in the HS group. Differential proteins including cytoskeletal and calcium-handling proteins, glycolytic enzymes, and oxidoreductase were mainly involved in the regulation of muscle contraction and energy metabolism that might together mediate meat quality development. Overall, this study provides a novel understanding about the mechanism of meat quality alterations as a result of pre-slaughter transport stress.