It has been shown that whole grains and dietary fiber are important for their fermentation characteristics in the large intestine, drawing more and more attention to quinoa and quinoa polysaccharides. In this study, we evaluated the prebiotic effect of quinoa seeds and quinoa polysaccharides after human simulated digestion. The modulatory effect of the quinoa and quinoa polysaccharides (QPs) on the gut microbiota was evaluated by the in vitro fermentation using human fecal microbiota. The yield of polysaccharides extraction was 15.45%. The digestibility of the cooked and uncooked quinoa after simulation of human digestion was 69.04% and 64.09%, respectively. The effect on the microbiota composition and their metabolic products was determined by the assessment of pH, short‐chain fatty acids (SCFAs), and changes in the bacterial population. After 24 hr anaerobic incubation, the total SCFAs of cooked, uncooked quinoa, and quinoa polysaccharides were 82.99, 77.11, and 82.73 mM, respectively with a pH decrease. At the phylum, genus, and class level, it has been found that the quinoa substrates enhance the growth of certain beneficial bacteria such as Prevotella and Bacteroides. Quinoa polysaccharides can be considered prebiotic due to their ability to increase Bifidobacterium and Collinsella. Principal component analysis (PCA) showed that there was a distinct modulating effect on the fecal microbiota which represents different distribution. Our research suggests that quinoa and quinoa polysaccharides have a prebiotic potential due to their association with the positive shifts in microbiota composition and short‐chain fatty acids production, which highlights the importance of further studies around this topic.