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Given the important roles of L-glutamine (L-Gln) in protein synthesis and energy metabolism, we hypothesized that L-Gln supplementation could improve growth performance and carcass characteristics in Hanwoo heifers. This study aimed to investigate the effects of L-Gln supplementation on growth performance, carcass characteristics, and gene expression in the liver and skeletal muscle of Hanwoo heifers. A total of 44 Hanwoo heifers (age: 23.7 ± 0.7 mo; body weight [BW]: 458 ± 68 kg) were allocated to three dietary treatment groups using a randomized block design, with the experimental start date as the blocking factor. The dietary treatments included L-Gln supplementation through top dressing based on the dry matter feed at 0% (n = 13), 0.25% (n = 16), and 0.5% (n = 15). The treatment lasted for 195 d (6.5 mo), followed by slaughter. Hematological parameters, growth performance, carcass characteristics, and gene expression in the liver and skeletal muscle samples obtained from the carcass were evaluated. Statistical analyses were performed using the MIXED procedure of SAS 9.4, with L-Gln supplementation as a fixed effect and block as a random effect. The 0.25% L-Gln group showed a significant increase (P < 0.05) in final BW, carcass weight, ribeye area, and fat color compared with the 0% group. In the liver, the expression of glutaminase 2 (GLS2) was significantly decreased (P < 0.05) in both the 0.25% and 0.5% L-Gln groups. Furthermore, the 0.5% L-Gln group exhibited lower (P < 0.05) expression levels of pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase 1 (PCK1), and phosphoenolpyruvate carboxykinase 2 (PCK2). The expression of glucose-6-phosphatase catalytic subunit 1 (G6PC1) also decreased (P < 0.05) in the 0.25% L-Gln group. In the skeletal muscle, myogenic differentiation 1 (MYOD) expression was significantly higher in the 0.25% L-Gln group, while the 0.5% L-Gln group showed higher zinc finger protein 423 (ZNF423) expression (P < 0.05). Additionally, the expression levels of myogenin (MYOG), heat shock protein beta-1 (HSPB1), CCAAT enhancer binding protein alpha (CEBPA), and fatty acid binding protein 4 (FABP4) were increased (P < 0.05) in both the 0.25% and 0.5% L-Gln groups. Overall, dietary supplementation with 0.25% L-Gln improved the final BW, carcass weight, and ribeye area in fattening Hanwoo heifers, which is possibly related to the regulation of myogenic genes, HSPB1, and enzymes involved in hepatic Gln metabolism and gluconeogenesis.