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Insufficient intake of n-3 polyunsaturated fatty acids is an important issue in modern Western-style diets. A large amount of evidence now suggests that a balanced intestinal microecology is considered an important part of health. Our results show that α-linolenic acid administration significantly improved the host metabolic phenotype and gut microbiota of mice fed a high-fat diet, and there was a correlation between the improved gut microbiota and metabolic phenotype. Some specific bacteria may play a unique regulatory role. Here, we have established correlation networks between gut microbiota and multitissue homeostasis, which may provide a new basis for further elucidating the relationship between the gut microbiota and host metabolism., Previous studies have shown that α-linolenic acid (ALA) has a significant regulatory effect on related disorders induced by high-fat diets (HFDs), but little is known regarding the correlation between the gut microbiota and disease-related multitissue homeostasis. We systematically investigated the effects of ALA on the body composition, glucose homeostasis, hyperlipidemia, metabolic endotoxemia and systemic inflammation, white adipose tissue (WAT) homeostasis, liver homeostasis, intestinal homeostasis, and gut microbiota of mice fed an HFD (HFD mice). We found that ALA improved HFD-induced multitissue metabolic disorders and gut microbiota disorders to various degrees. Importantly, we established a complex but clear network between the gut microbiota and host parameters. Several specific differential bacteria were significantly associated with improved host parameters. Rikenellaceae_RC9_gut_group and Parasutterella were positively correlated with HFD-induced “harmful indicators” and negatively correlated with “beneficial indicators.” Intriguingly, Bilophila showed a strong negative correlation with HFD-induced multitissue metabolic disorders and a significant positive correlation with most beneficial indicators, which is different from its previous characterization as a “potentially harmful genus.” Turicibacter might be the key beneficial bacterium for ALA-improved metabolic endotoxemia, while Blautia might play an important role in ALA-improved gut barrier integrity and anti-inflammatory effects. The results suggested that the gut microbiota, especially some specific bacteria, played an important role in the process of ALA-improved multitissue homeostasis in HFD mice, and different bacteria might have different divisions of regulation. IMPORTANCE Insufficient intake of n-3 polyunsaturated fatty acids is an important issue in modern Western-style diets. A large amount of evidence now suggests that a balanced intestinal microecology is considered an important part of health. Our results show that α-linolenic acid administration significantly improved the host metabolic phenotype and gut microbiota of mice fed a high-fat diet, and there was a correlation between the improved gut microbiota and metabolic phenotype. Some specific bacteria may play a unique regulatory role. Here, we have established correlation networks between gut microbiota and multitissue homeostasis, which may provide a new basis for further elucidating the relationship between the gut microbiota and host metabolism.