Cats with chronic kidney disease (CKD) have a decreased ability to maintain body weight. As CKD advances, loss of body weight contributes to morbidity and mortality. The goal of this study was to evaluate the combined effects of feeding betaine and prebiotics on body weight of both CKD and healthy cats. The pre-trial food (control food) was a complete and balanced dry food designed to aid in the management of CKD. Test food was the control food supplemented with betaine (0.500%) and prebiotics: long-chain oat beta-glucan (0.586%) and 0.407% short chain fructooligosaccharides (scFOS). The CKD cats (n = 7) were fed pre-trial food for 28 days and then randomly assigned to control food or test food. Each food was fed for 8 weeks in a cross-over study design. In a second study, healthy cats received control food or test food for 8 weeks (n = 8 each group). Blood, urine, and fecal samples were collected to evaluate concentrations of relevant kidney function biomarkers and metabolites at the end of each feeding period for CKD cats, and blood samples were collected monthly to evaluate concentrations of plasma metabolites for healthy cats. Body weight and composition were measured using dual-energy X-ray absorptiometry (DEXA) scan at baseline and after each feeding period. Total body mass was significantly higher in CKD cats after consuming test food compared with control food (P = 0.004), with no significant difference in food intake while consuming test or control food (P = 0.34). Test food did not affect total body mass or composition of healthy cats. Indole compounds produced by bacterial metabolism were decreased in urine and increased in feces of CKD cats fed test food, and plasma concentrations were negatively correlated with the level of kidney function, indicating a potential benefit of consuming test food. In healthy cats, consuming test food resulted in significantly decreased concentrations of plasma P-cresol sulfate (P = 0.004) and increased concentrations of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA; both P < 0.05), despite the fact that both control and test foods had similar concentrations of these long-chain fatty acids, 0.03% and 0.02%, respectively. These results suggest that the addition of betaine and prebiotics to the control food formula may have increased total body mass in CKD cats by enhancing one-carbon metabolism and by modulating the gut microbiome.