Goji berries show health benefits, although the possible mechanisms of action, including compositional changes in the gut microbiome, are still not fully understood. The aim of this study was to evaluate the effect of Goji berry supplementation on microbiota composition and metabolites in the digestive tracts of rabbits. Twenty-eight New Zealand White rabbits were fed with a commercial feed (control group, C; n = 14) or the same diet supplemented with 3% of Goji berries (Goji group, G; n = 14), from weaning (35 days old) until slaughter (90 days old). At slaughter, samples from the content of the gastrointestinal tracts were collected and analyzed by Next Generation 16S rRNA Gene Sequencing to evaluate the microbial composition. Ammonia and lactic acid were also quantified in caecum. Results showed differences in microbiota composition between the groups for two phyla (Cyanobacteria and Euryarchaeota), two classes (Methanobacteria and Bacilli), five orders, fourteen families, and forty-five genera. Ruminococcaceae (p < 0.05) and Lachnospiraceae (p < 0.01) were more abundant in G than in C group. Lactobacillaceae also showed differences between the two groups, with Lactobacillus as the predominant genus (p = 0.002). Finally, Goji berry supplementation stimulated lactic acid fermentation (p < 0.05). Thus, Goji berry supplementation could modulate gastrointestinal microbiota composition and caecal fermentation.