High-fat diet (HFD)-induced obesity has been associated with alteration of gut microbiota alongside body weight gain. In this study, the synbiotic effect of Lactobacillus gasseri 505 (LG) and Cudrania tricuspidata (CT) in HFD-induced mice was revealed. After feeding mice with high-fat diet for 10 weeks, combination of LG and CT (LG_CT) exhibited the greatest reduction in the final body weight (11.9%). Moreover, microbial diversity significantly increased, and Principal Coordinate Analysis (PCoA) revealed that the LG_CT group showed closer cluster to NORM. At phylum level, the Firmicutes/Bacteroidetes (F/B) ratio increased in HFD, and the abundance of Bacteroidetes was restored by LG and CT. At genus level, notable changes in Alistipes, Desulfovibrio, Bilophila, and Acetatifactor were observed. Helicobacter elevated to 16.2% in HFD and diminished dramatically to less than 0.01% in LG and/or CT. At species level, L. gasseri increased after the administration of LG (0.54%) and LG_CT (1.14%), suggesting that LG may grow and colonize in the gut and CT can function as a prebiotic. Finally, functional analysis revealed certain metabolic factors correlated with body weight and gut microbiota. This study serves as a potential basis for the application of L. gasseri 505 and C. tricuspidata in the prevention and treatment of diet-induced obesity.