Introduction. Obesity is an inflammatory condition associated with metabolic alterations. Recent researches suggested that gut microbiota plays a role in its pathogenesis, as obesity has been associated with lower bacterial diversity and higher Firmicutes/Bacteroidetes ratio (F/R ratio) compared to normal‐weight condition. Such alterations can affect metabolite production (mainly short-chain fatty acids) having an impact on inflammatory markers modulation and insulin secretion. Objectives and study. To determine the effect of 4 months 500 mg/day DHA supplementation, combined with dietary and lifestyle intervention, on gut microbiota and biochemical parameters. Methods. Twelve Caucasian obese children (5 males, 7 females), aged 6-14 years (mean age 10 y), were enrolled. Study protocol was approved by the local Ethics Commitee and registered on ClinicalTrials.gov (identifier: NCT04151758). Blood and stool samples were collected at baseline (t0), after 4 months DHA supplementation combined with diet-lifestyle intervention (t1), and after additional 4 months of dietary dietary-lifestyle intervention without DHA supplementation (t2). Anthropometric measures (BMI z score, body circumferences and skinfolds), body composition estimation using air displacement plethysmography system (BOD POD), and 3-day food record were assessed. Biochemical analysis included: glucose metabolism, lipid profile, liver function and inflammatory markers. Insulin sensitivity and insulin resistance were assessed by HOMA index (Homeostasis Model Assessment) and QUICKI (Quantitative Insulin-Sensitivity Check Index), respectively, while cardiovascular risk was estimated trough AIP (Atherogenix Index of Plasma). Gut microbiota analysis was performed by next-generation sequencing using V3–V4 hypervariable 16S rRNA genomic region (Illumina). Results. So far 12 children completed the DHA plus dietary-lifestyle intervention (t0-t1-t2) and 3, out of these 12, completed the entire observation period (t0-t1-t2). In both groups no longitudinal changes in dietary habits, as well as in BMI z-score, waist circumference and fat mass percentage (FM%) were recorded. At baseline, most patients showed increased values of HOMA-IR (Mean value 3.30, SD 1.92), AIP (0.24, SD 0.22), VES (26.75 mm/h, SD 12.4) and CPR (6.80 mg/dl, SD 2.97), suggesting different grades of insulin resistance, dyslipidemia and systemic inflammation. No significant improvement was found after the intervention (t1). Alanine transferase values were slightly reduced from t0 (34.33, SD 5.69 IU/L) to t1 (31.33, SD 11.68 IU/L) and difference became statistically significant from t0 to t2 (p = 0.014, 3 subjects). While alpha-diversity analysis revealed no significant differences in gut microbiota biodiversity at different time-points, phylogenetic analysis highlighted a significant separation of t0 and t1 bacterial communities according to weighted Unifrac distances (p = 0.017). In particular, a significant lowering of F/R ratio from t0 (4.04) to t1 (2.20) was observed. Conclusions. Lowering of F/R ratio from t0 to t1 seems to be associated exclusively to DHA supplementation. The study is still on-going and more data from t2 time-point, as well as microbial metabolite analysis, are needed to support the potential impact of DHA supplementation.