Changes in the composition of gut microbiota have been proposed as possible causes of obesity. Our previous study showed that glutamic acid (Glu) alone or a combination of Glu and arginine (Arg) decreased backfat thickness in finishing pigs. Therefore, this study aimed to investigate the effects of dietary supplementation with Glu and/or Arg on body fat weight, composition of gut microbiota, and short-chain fatty acid concentrations in the colons of finishing pigs, and to elucidate whether body fat weight was associated with changes in the colonic microbial community and concentrations of colonic metabolites. Sixty Duroc × Large White × Landrace pigs with an average initial body weight of 77.1 ± 1.3 kg were randomly assigned to one of five treatment groups (12 pigs per group). The pigs in the control group were fed a basal diet (BD group), while those in the experimental groups were fed a basal diet supplemented with either 2.05% l-alanine (Isonitrogenous, IS group), 1.00% l-arginine (Arg group), 1.00% glutamate + 1.44% l-alanine (Glu group), or 1.00% l-arginine + 1.00% glutamate (Arg_Glu group). The results showed that dietary supplementation with alanine has no effect (P > 0.05) on body fat weight, while with both Glu and Arg + Glu decreased (P < 0.05) body fat weight and increased (P < 0.05) colonic concentrations of propionate, butyrate, and valerate relative to the BD group. Negative relationships (P < 0.05) were observed between body fat weight and colonic propionate and butyrate concentrations. Compared to the IS group, dietary supplementation with Arg or Arg + Glu decreased (P < 0.05) colonic tyramine concentration. Compared to the IS group, the butyrate concentration increased (P < 0.05) in the Arg_Glu group. Compared to the BD group, the relative colonic abundance of Actinobacteria was higher (P < 0.05) in the Arg_Glu group. The abundances of Lachnospiraceae_XPB1014_group, norank_f_Erysipelotrichaceae, and Roseburia sp. were negatively (P < 0.05) correlated with body fat weight, and the abundance of norank_f_Erysipelotrichaceae was also negatively (P < 0.05) correlated with colonic butyrate concentration. These findings suggest that decreased body fat weight in finishing pigs can be induced by Glu supplementation alone or in combination with Arg. Glu + Arg supplementation was also associated with increased colonic butyrate and propionate concentrations and increased colonic Actinobacteria abundance.