Cold stress disrupts broiler homeostasis, and a single intervention may be insufficient for protection. This study examined the effects of early cold conditioning (25 ± 1 °C for 3 h at 5 days) and glutamine (Gln) supplementation on broiler thermotolerance during a 16 ± 1 °C cold challenge at 35 days. A 2 × 3 factorial design assigned 360 Cobb-500 broilers to six treatments (six replicates/treatment, ten birds/replicate) with three Gln levels (0%, 0.3%, and 0.5%) and two temperature conditions: standard temperature and a 7 °C reduction at 5 days old. Supplementing with 0.3% and 0.5% Gln and cold conditioning improved growth performance (p < 0.05), except for feed intake during the grower-finisher phase and overall growth with cold conditioning alone. Adding 0.3% and 0.5% Gln enhanced hemoglobin, total protein, albumin, triiodothyronine (T3), thyroxine (T4), antioxidant capacity, catalase (CAT), superoxide dismutase (SOD), heat shock protein 70 (HSP70), interleukin 2 (IL2), IL10, IL4, interferon-γ (INF-γ), and troponin-T levels (p < 0.05). Cold conditioning influenced packed cell volume, T3, T4, CAT, HSP70, IL10, INF-γ, and troponin-T levels (p < 0.05). Diet-temperature interaction influenced growth, antioxidant, and immune responses, but not hematological or biochemical indicators. Overall, dietary Gln at 0.3% and 0.5% and early cold conditioning may serve as complementary strategies to mitigate cold stress in broilers.