Abstract: The intestinal propionic acid level in patients with methylmalonic acidemia (MMA) may affect the course of the disease, but the current intervention strategy is still unclear. This study aimed to elucidate the differences in fecal propionic acid levels and gut microbiota composition between patients with MMA and healthy people, as well as to identify dietary factor combinations that effectively reduce propionic acid production through in vitro fecal fermentation model and MMA rat model. This investigation provides a novel strategy for dietary management in patients with MMA. Gas chromatography-mass spectrometry and metagenomic sequencing were employed to analyze propionic acid content, gut microbiota structure, and species composition in the feces of 28 patients with MMA and 17 healthy people. Subsequently, an in vitro fecal bacterial fermentation model was utilized to screen for optimal combinations among 12 dietary factors capable of significantly reducing propionic acid levels. Finally, 5-day-old Wistar rats (segregated into male and female subgroups) were selected to construct the MMA model and randomly divided into five groups: control group, model group, galactooligosaccharide+arabinogalactan group (GOS+AG group), galactooligosaccharide+tremella polysaccharide group (GOS+TPs group), and arabinogalactan+tremella polysaccharide group (AG+TPs group). Propionic acid content in rat feces was measured and analyzed at three time points: prior to intervention (14 days), mid-stage (21 days), and late stage (28 days). The results demonstrated significant differences in fecal propionic acid levels (P<0.001) and gut microbiota structure (P=0.003) between patients with MMA and healthy people. Compared to the control group, higher abundances of Blautia wexlerae, Bacteroides thetaiotaomicron, and Blautia producta in the MMA group may represent critical factors influencing changes in intestinal propionic acid levels. After 24 hours of fermentation, screening identified that poria cocos polysaccharides, fructooligosaccharides, galactooligosaccharides, arabinogalactan, and tremella polysaccharides demonstrated significant efficacy in reducing propionic acid levels (P<0.05). Following the combination of the aforementioned five polysaccharides and subsequent fermentation, three combinations with the most effective reduction were identified: GOS+AG group, GOS+TPs group, AG+TPs group (P<0.001). Ultimately, animal experiments confirmed that the combination of AG+TPs group yielded the best results (P<0.0001). In conclusion, differences in fecal propionic acid levels between patients with MMA and healthy people may be associated with variations in gut microbiota composition. The combination of AG+TPs group can effectively reduce propionic acid levels, offering a promising dietary management strategy for patients with MMA.