ABSTRACT: Adherence to Mediterranean diet (MD) and physical activity (PA) in adolescence represent powerful indicators of healthy lifestyles in adulthood. The aim of this longitudinal study was to investigate the impact of nutrition education program (NEP) on the adherence to the MD and on the inflammatory status in healthy adolescents, categorized into three groups according to their level of PA (inactivity, moderate intensity, and vigorous intensity). As a part of the DIMENU (Dieta Mediterranea & Nuoto) study, 85 adolescents (aged 14-17 years) participated in the nutrition education sessions provided by a team of nutritionists and endocrinologists at T0. All participants underwent anthropometric measurements, bio-impedentiometric analysis (BIA), and measurements of inflammatory biomarkers such as ferritin, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels. Data were collected at baseline (T0) and 6 months after NEP (T1). To assess the adherence to the MD, we used KIDMED score. In our adolescents, we found an average MD adherence, which was increased at T1 compared with T0 (T0: 6.03 ± 2.33 vs. T1: 6.96 ± 2.03, p = 0.002), with an enhanced percentage of adolescents with optimal (≥8 score) MD adherence over the study period (T0: 24.71% vs. T1: 43.52%, p = 0.001). Interestingly, in linear mixed-effects models, we found that NEP and vigorous-intensity PA levels independently influenced KIDMED score (β = 0.868, p < 0.0001 and β = 1.567, p = 0.009, respectively). Using ANOVA, NEP had significant effects on serum ferritin levels (p < 0.001), while either NEP or PA influenced ESR (p = 0.035 and 0.002, respectively). We also observed in linear mixed-effects models that NEP had a negative effect on ferritin and CRP (β = -14.763, p < 0.001 and β = -0.714, p = 0.02, respectively). Our results suggest the usefulness to promote healthy lifestyle, including either nutrition education interventions, or PA to improve MD adherence and to impact the inflammatory status in adolescence as a strategy for the prevention of chronic non-communicable diseases over the entire lifespan.