The incidence of feline obesity continues to rise despite it being a preventable disease. There are many risks and health perturbations associated with obesity, with several of those impacting a pet’s quality of life, wellness, and longevity. Feline obesity is commonly studied, but most research has been focused on weight loss rather than weight gain. To our knowledge, feline studies have not examined the implications of overfeeding and weight gain on gastrointestinal transit time (GTT) nor the association it has with the fecal microbiota. Therefore, the objective of this study was to determine the effects of overfeeding and weight gain on apparent total tract digestibility (ATTD), GTT, blood hormones, serum metabolites, hematology, fecal microbiota populations, and voluntary physical activity of cats. Eleven lean adult spayed female cats [body weight (BW) = 4.11 ± 0.43 kg; body condition score (BCS) = 5.41 ± 0.3; age = 5.22 ± 0.03 y] were used in a longitudinal weight gain study. After a 2-wk baseline phase, cats were allowed to overeat for 18 wk. A commercially available complete and balanced diet was fed during the baseline phase to identify intake needed to maintain BW. Cats were then fed the same diet ad libitum to induce weight gain. Fecal samples, blood samples, and voluntary physical activity data were collected at baseline (wk 0) and 6, 12, and 18 wk after weight gain. Fecal samples were collected for microbiota analysis, determination of ATTD, and GTT measurement while blood samples were collected for serum chemistry, hematology, and insulin and leptin measurements. Microbiota data were evaluated using QIIME2. All other measures were evaluated statistically using the Mixed Models procedure of SAS using repeated measures analysis, with time effects being the focus. A P<0.05 was considered significant. The ATTD of dry matter (P=0.0061), organic matter (P=0.0130), crude protein (P<0.0001), fat (P=0.0002) and gross energy (P=0.0002) and GTT (P=0.0418) decreased with overfeeding and weight gain. Fecal bacterial alpha diversity measures were unchanged, but fecal bacterial beta diversity was impacted (P<0.05) with overfeeding and weight gain. The relative abundances of 16 bacterial genera, including Bifidobacterium, Collinsella, Erysipelatoclostridium were affected (P<0.05) by overfeeding and weight gain. In conclusion, overfeeding and subsequent weight gain reduced ATTD, reduced GTT, and caused changes to the fecal microbial community of adult cats.