Green-plant thylakoids increase satiety by affecting appetite hormones such as ghrelin, cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1). The objective of this study was to investigate if thylakoids also affect gastrointestinal (GI) passage and microbial composition. To analyse the effects on GI passage, 16 rats were gavage-fed a control or thylakoid-supplemented high-fat diet (HFD) 30 min before receiving Evans blue. Another 16 rats were fed a control HFD or thylakoid HFD for two weeks prior to the intragastric challenge with Evans blue. The amount of Evans blue in the stomach and the distance of migration in the intestines after 30 min were used as a measurement of gastric emptying and intestinal transit. These were reduced by thylakoid supplementation in the acute study, and however not significantly also after the two-week diet study. The second aim of the study was to investigate if thylakoid-supplementation affects the gut microbiota and amount of faecal fat in healthy human volunteers (n = 34) receiving thylakoid or placebo treatments for three months. Microbiota was analysed using 16S rRNA gene sequencing and qPCR, and faecal fat was extracted by dichloromethane. The total bacteria, and specifically the Bacteriodes fragilis group, were increased by thylakoid treatment versus placebo, while thylakoids did not cause steatorrhea. Dietary supplementation with thylakoids thus affects satiety both via appetite hormones and GI fullness, and affects the microbial composition without causing GI adverse effects such as steatorrhea. This suggests thylakoids as a novel agent in prevention and treatment of obesity.