Optimising nutritional strategies to enhance meat quality and animal welfare remains a major challenge in modern pig production. Early postweaning stress often impairs growth, muscle development, and immune responses, which can negatively affect carcass traits and pork quality. Among various nutritional interventions, amino acid supplementation has gained attention, with glutamine (Gln) considered particularly important due to its roles in metabolism, immunity, and muscle physiology. The present study aimed to investigate the effects of dietary Gln supplementation on the carcass traits, proximate composition, amino acid profile, inflammatory markers, and the most potent antioxidant in three different muscles (triceps brachii, longissimus lumborum - LL, and semimembranosus) in weaner gilts and barrows. The study involved 24 crossbred pigs, half barrows and half gilts, divided into two groups: one supplemented with 0.5% L-glutamine (0.5% Gln) and a control group. After a 42-day supplementation period just after weaning, pigs were slaughtered when weighing, on average, 123.77 kg, and muscle samples were collected for analysis. Results indicated that Gln supplementation significantly influenced carcass traits, including increased hot carcass weights and leanness, particularly notable in barrows. Proximate composition analysis revealed varied effects of Gln supplementation on protein and fat content across different muscles and pig sexes. Gln supplementation generally increased essential amino acid concentrations (especially in LL), with significant variations observed between sexes. Moreover, supplementation led to reduced concentrations of pro-inflammatory interleukin-1 β (IL-1β) and tumour necrosis factor α (TNF-α) in certain muscles, indicating potential anti-inflammatory effects. However, there were no significant differences in instrumentally assessed muscle colour. These findings suggest that dietary supplementation with Gln can enhance pork’s nutritional quality by improving protein composition and reducing muscle inflammatory markers. By enhancing essential amino acid concentrations and mitigating pro-inflammatory cytokines, Gln supplementation shows promise not only in improving meat quality but also in promoting animal health and welfare.