Cotton production faces significant challenges from insect pests, with chemical pesticide use becoming increasingly limited by resistance and environmental concerns. This study explores the potential use of caffeine, a natural plant alkaloid, as an environmentally friendly insect resistance strategy in cotton. Exogenous caffeine application demonstrated potent insecticidal effects against cotton bollworm (Helicoverpa armigera) larvae, with concentrations ≥ 2 mg mL⁻¹ causing near-complete feeding cessation and up to 70% larval mortality. Building on this, we engineered transgenic cotton (Gossypium hirsutum cv. Jin668) for heterologous caffeine biosynthesis by introducing three key N-methyltransferase genes (CaXMT1, CaMXMT1, CaDXMT1) by multiple gene transformation. Transgenic lines expressing all three genes showed remarkable caffeine accumulation (up to 3.59 mg g⁻¹ dry weight), whereas two-gene combinations exhibited wild-type-level production. Feeding preference assays revealed that caffeine-enriched cotton strongly deterred feeding by H. armigera. Non-choice feeding trials demonstrated reduced leaf consumption and reduced larval growth in H. armigera fed on caffeine-producing cotton. The study highlights the effectiveness of synthetic biology approaches using the TGSII-UNiE multigene stacking system, despite challenges in transgene stability. This work advances plant-derived insect resistance research and provides a sustainable framework for reducing chemical pesticide reliance in cotton production, while underscoring unique potential of cotton as a synthetic biology platform for secondary metabolite engineering.