Recent research advances examining the gut microbiome and its association with human health have indicated that microbiota-targeted intervention is a promising means for health modulation. In this study, elderly people in long-term care (aged 83.2 ± 5.3 year) with malnutrition (MNA-SF score ≤ 7) were recruited in a community hospital for a 12-week randomized, single-blind clinical trial with Clostridium butyricum. Compared with the basal fluctuations of the control group, an altered gut microbiome was observed in the intervention group, with increased (p < 0.05) Coprobacillus species, Carnobacterium divergens, and Corynebacterium_massiliense, and the promoted growth of the beneficial organisms Akketmanse muciniphila and Alistipes putredinis. A concentrated profile of 14 increased Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs (KOs) that were enriched in cofactor/vitamin production and carbohydrate metabolism pathways were discovered; the genes were found to be correlated (p < 0.05) with an elevated abundance of plasma metabolites and short-chain fatty acids (SCFAs), unsaturated medium- to long-chain fatty acids (MFA, LFA), carnitines, and amino acids, thus suggesting a coordinated ameliorated metabolism. Proinflammatory factor interferon-gamma (IFN-γ) levels decreased (p < 0.05) throughout the intervention, while the gut barrier tight junction protein, occludin, rose in abundance (p = 0.059), and the sensitive nutrition biomarker prealbumin improved, in contrast to the opposite changes in control. Based on our results obtained during a relatively short intervention time, C. butyricum might have great potential for improving nutrition and immunity in elderly people in long-term care with malnutrition through the alteration of gut microbiota, increasing the abundance of beneficial bacteria and activating the metabolism in SCFA and cofactor/vitamin production, bile acid metabolism, along with efficient energy generation.