In many Iranian EFL classrooms, vocabulary instruction still depends largely on teacher-centered and traditional teaching practices. In these contexts, principles associated with Neuroscience-Informed Instruction (NII) are also rarely incorporated. This situation has increased interest in instructional approaches that are more consistent with how learners process and recall information. Accordingly, this study investigated the effects of NII on vocabulary development and long-term retention among Iranian EFL learners. Twenty-eight intermediate EFL learners (18–31 years old) from a private language institute in Kerman, Iran were selected through the Oxford Placement Test (OPT) and assigned to two intact classes. The experimental group received instruction based on NII principles, including spaced repetition, retrieval practice, multimodal input, emotional engagement, and brief metacognitive reflection. The control group received conventional vocabulary instruction. The intervention was carried out over twelve 90-minute sessions across six weeks. A researcher-made test measured vocabulary knowledge through a pre-test, immediate post-test, and delayed post-test. The data were analyzed using independent-samples t-tests and mixed-design repeated-measures ANOVA. Both groups improved over time; however, the experimental group achieved higher scores on both the immediate and delayed post-tests. The repeated-measures analyses also revealed a statistically significant Time × Group interaction, with the experimental group demonstrating stronger vocabulary growth and retention over time. These findings suggest that instructional practices informed by cognitive neuroscience principles may support more effective vocabulary learning and retention in EFL contexts. The findings highlight the potential value of neuroscience-informed instructional practices for vocabulary teaching in EFL contexts.