In humans, gastrointestinal microbiota of obese subjects harbor a less diverse bacterial community and a lower ratio of Bacteriodetes:Firmicutes phyla. However, these relationships have not been examined in the domestic cat. Thus, the objective of this study was to characterize the hindgut microbiota of domestic cats transitioning from a lean to an obese phenotype. Nine domestic male cats (mean age = 8 ± 0.3 yr; mean BW = 4.5 ± 0.4 kg, mean BCS range = 5.0 ± 0.5) were ad libitum fed a complete and balanced experimental diet for 36 wk. Food intake was measured daily, while BW and BCS were measured weekly, fresh fecal samples were collected at 0, 6, 12, 24 and 36 wk. Amplification of fecal bacterial DNA was performed using targeted primers and a Fluidigm Access Array. Illumina sequencing was performed utilizing a MiSeq2000 using v3 reagents. High quality (quality value >25) sequence data derived from the sequencing process were analyzed using QIIME 1.8.0. Resulting sequences were clustered into operational taxonomic units (OTU) using closed-reference OTU picking against the Greengenes 13_8 reference OTU using a 97% similarity threshold. Data were analyzed as repeated measurements, with post-hoc Tukey adjustment. A P < 0.05 was considered statistically significant. A total of 9,145,663 reads were generated in the current data set, with an average of 203,236 reads per sample used to provide diversity estimates. Cats incrementally changed from a lean phenotype (mean BCS = 5.0) at baseline to an overweight phenotype with a mean BCS of 7.5 at 36 wk. Similar to previously observed in humans, overweight cats had a lower ratio of Bacteriodetes (e.g., Bacteroides; Prevotella) to Firmicutes (e.g., Lactobacillus) phyla. However, microbial diversity and species richness was not altered by BW gain (~250 species). Body weight gain resulted in a gut microbiota shift depicted by unweighted UniFrac-based PCoA analysis between cats at baseline and 6 wk vs. 24 and 36 wk. Proteobacteria (e.g., Succinivibrionaceae) and Fusobacteria (Fusobacteriaceae) were decreased (P < 0.05) by BW gain. These data demonstrate the transition from a lean to an overweight phenotype alters the GI microbiota in cats.