Evidence suggests that plant-derived nanovesicles may play a significant role in human health. Tartary buckwheat has several physiological activities; however, its underlying health-promoting mechanism remains unclear. In this study, first, Tartary buckwheat-derived nanovesicles (TBDNs) were collected, their structures were analyzed, and microRNA sequencing was performed. Next, target prediction and functional verification were conducted. Finally, the effects of TBDNs on gut microbiota and short-chain fatty acid levels were evaluated. The average size of TBDNs was 141.8 nm diameter. Through the sequencing analyses, 129 microRNAs, including 11 novel microRNAs were identified. Target gene prediction showed that some microRNAs could target functional genes in Escherichia coli and Lactobacillus rhamnosus-related physiological processes. TBDNs significantly promoted the growth of E. coli and L. rhamnosus, enhanced the diversity of fecal microorganisms and increased the short-chain fatty acid levels. These findings provided a new nutritional perspective for Tartary buckwheat and were conducive to promote the development and utilization of Tartary buckwheat.

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