Charcoal-grilled food is favored for its strong roasted and nutty aroma, and grilled lamb’s flavor characteristics are significantly affected by compounds such as 2,5-dimethylpyrazine (2,5-DMP). This study explored the formation and reactivity of 2,5-DMP in the grilled lamb using food and model reaction systems integrated with density functional theory (DFT). Grilling experiments were performed at (180 ± 5) °C with controlled time intervals (0–30 min), while DFT calculations employed the 6-311G (d,p) basis set to analyze molecular interactions. The results showed that a total of 36 aroma compounds were identified during grilling. The 2,5-DMP concentration peaked at 25 min. The model reaction systems confirmed glycine and glucose as primary precursors, producing 339.51 ng of 2,5-DMP. The DFT analysis identified glucose’s C5–OH group and glycine’s –NH2 group as key reactive sites, facilitating the nucleophilic and electrophilic interactions. It revealed low activation energy and high reactivity, elucidating the reaction pathway involving nucleophilic substitution, dehydration, and condensation. This study provides novel insights into 2,5-DMP formation mechanisms and offers a foundation for optimizing grilling processes to enhance the aroma and sensory quality of meat products.