Display omitted •Polysaccharides from Gracilaria chouae (GCPs) were prepared using acid & water extraction.•Digestion & fermentation properties of GCPs were evaluated both in vitro and in vivo.•Acid extracted GCP and inulin showed similar microbial fermentation properties.•Acid extraction for polysaccharides could be a promising way to obtain new prebiotics. The sulfated polysaccharides extracted from Gracilaria chouae using critic acid extraction and water extraction, respectively, and their digestion and fermentation characteristics were compared in vitro and in vivo. The molecular weight of water extracted polysaccharide of G. chouae (WGCP) was 1.73 × 103 kDa while critic acid extracted polysaccharide (CGCP) was 31.5 kDa. During stimulated gastrointestinal digestion in vitro, WGCP and CGCP were lightly degraded. However, the glycemic index (GI) of WGCP and CGCP were 17.7 and 36.12, respectively. After 24 h of fermentation in vitro, the pH values of CGLP in the fecal culture decreased from 6.89 to 4.82, similar to the inulin but significantly (p < 0.05) lower than those of the WGCP and normal control. In addition, CGCP and inulin showed similar microbial fermentation characteristics according to the microbiome compositions and contents of short-chain fatty acids (SCFAs). Nevertheless, CGCP gavage for four weeks could also promote the growth of microbes producing the SCFAs such as Peptococcus, Roseburia and Butyricicoccus in the cecum of KM mice. The present study suggests that polysaccharides prepared by acid-extraction method could potentially be used as a good source of prebiotics.