Byline: Xin Yan, Chengfeng Yang, Guopeng Lin, Yuqing Chen, Song Miao, Bin Liu,Chao Zhao Keywords: antidiabetic; Enteromorpha prolifera; flavonoids; gut microflora; insulin signaling Abstract This study aimed to investigate the antidiabetic activity of water-ethanol extract of green macroalgae Enteromorpha prolifera (EPW) and its flavonoid-rich fraction less than 3 kDa (EPW3) in type 2 diabetic mice induced by streptozotocin and a high-sucrose/high-fat diet. The major active compounds were identified using ultra-performance liquid chromatography coupled with quadrupole-time-of-flight-tandem mass spectrometry. Quantitative gene expression analysis of the insulin signaling pathway was performed. The effects of EPW3 on gut microflora in diabetic mice were analyzed by high-throughput 16S rRNA gene sequencing. The results showed EPW3 treatment decreased the fasting blood glucose, improved oral glucose tolerance, and protected against liver and kidney injury with reduced inflammation in diabetic mice. The active principle of EPW3 revealed hypoglycemic effect as indicated by activation of the IRS1/PI3K/AKT and inhibition of the JNK1/2 insulin pathway in liver. Furthermore, the treatment significantly enriched the abundance of Lachnospiraceae and Alisties, which were positive correlation of metabolic phenotypes. These findings indicated that EPW3 possessed great therapeutic potential as adjuvant therapy for type 2 diabetes. Article Note: Authors Xin Yan and Chengfeng Yang contributed equally to this study. CAPTION(S): Table S1-The histological score system for grading hepatitis activity. Table S2-Scoring systems for cortical tubules and interstitial fibrosis. Table S3-The primer sequences of target genes for q-PCR assay. Table S4-Characterization of major metabolites of EPW3 by UPLC-Q-TOF-MS/MS. Figure S1-Chromatographic peaks of EPW3 in UPLC. The graphic in the top right presented the spectrums of the blank. Figure S2-Representative UPLC-Q-TOF-MS/MS chromatographs of EPW3. Figure S3-Effects of EPW and EPW3 treatment on the level of protein expression of representative genes in liver of type 2 diabetic mice. Figure S4-Bacterial distribution of the abundant genus among these 4 groups. Double hierarchical dendrogram shows the bacterial distribution. The heatmap plot depicts the relative percentage of each bacterial genus within each group. The relative values for bacterial genus are indicated by color intensity with the legend indicated at the top right corner.