We investigated the influences of dietary rumen-protected guanidinoacetic acid (RPGAA) on the growth, slaughter performance, and meat quality characteristics of Simmental bulls. In this study, 56 bulls (615 ± 8.9 kg) were randomly assigned to 74-day treatment in four group: control, low-RPGAA (0.3 g/kg dry matter [DM]-guanidinoacetic acid [GAA]), medium-RPGAA (0.6 g/kg DM-GAA), and high-RPGAA (0.9 g/kg DM-GAA), with 14 bulls per group. With RPGAA supplementation, the average daily gain increased quadratically (P < 0.05), whereas the feed-conversion ratio decreased linearly (P = 0.027). Additionally, blood total protein and albumin levels increased linearly (P < 0.05), while creatine (Cr), creatine kinase, and insulin-like growth factor 1 increased quadratically (P < 0.05). RPGAA supplementation linearly increased (P < 0.05) dressing percentage, lean percentage, and loin eye area, while linearly decreasing (P < 0.05) backfat thickness, fat percentage and subcutaneous fat percentage. The pH at 24-h postmortem and shear force of the Longissimus lumborum muscle were linearly increased (P < 0.05), whereas cooking and drip losses were linearly reduced (P < 0.05). RPGAA supplementation linearly increased (P = 0.017) protein content; quadratically elevated (P = 0.021) ether extract content, the percentage of phosphorylated Cr/Cr, and the adenosine diphosphate/adenosine triphosphate ratio, while linearly decreasing moisture content (P = 0.012). Medium-RPGAA supplementation significantly increased (P < 0.05) protein expression related to muscle proliferation, development, protein synthesis, and intramuscular fat synthesis, while significantly decreasing (P < 0.05) proteins expression correlated with subcutaneous fat deposition. In conclusion, dietary RPGAA supplementation enhanced the growth, slaughter performance, and meat nutritional quality of fattened Simmental bulls by altering protein expression related to muscle proliferation, development, protein synthesis, subcutaneous fat deposition, and intramuscular fat synthesis.