Abstract: To understand the physicochemical properties of quinoa RS3-type resistant starch (RS3-type QRS) and the effects of its in vitro fermentation on the intestinal flora and short-chain fatty acids (SCFAs), quinoa starch (QS) was prepared via alkali extraction and then autoclaved to obtain RS3-type QRS, and the amylose content, transmittancy, pasting properties, and thermal properties of both starches were analyzed. The in vitro fermentation characteristics of the two and their effects on intestinal metabolites were further explored using an in vitro fecal simulation fermentation system, 16S rRNA sequencing, liquid chromatography-mass spectrometry, and non-targeted metabolomics. Compared with QS, RS3-type QRS had significantly higher contents of resistant starch and amylose (P<0.05), significantly improved transmittancy and solubility (P<0.05), possessed more stable thermodynamic properties, and was better in quality and easier to cook. After adding RS3-type QRS to the in vitro fermentation matrix, the abundance of Faecalibacterium increased whereas that of Escherichia-Shigella decreased. Members of the Firmicutes, including Blautia and Lactococcus, formed the core community in RS3-type QRS and were significantly positively correlated with butyric acid enrichment (P<0.01 or P<0.05). In metabolic pathways of bile secretion, the bile acid levels were significantly increased (P<0.05). SCFAs produced by the intestinal flora, together with the differential metabolites, maintained intestinal homeostasis. The findings from this study provide a reference for exploring the impact of RS3-type QRS on intestinal health and a theoretical basis for its application.