Carbapenem-resistant Klebsiella pneumoniae (CRKP) causes serious intensive care unit (ICU)-acquired infections, yet the mechanisms of microbiota-mediated colonization resistance remain unclear. We analyzed the gut microbiome and metabolic profiles of healthy individuals and ICU patients, distinguishing those with and without CRKP colonization. ICU patients showed distinct microbial communities compared to healthy controls, and CRKP-positive patients exhibited unique microbial and metabolic signatures. We demonstrated that a healthy gut microbiome is essential for providing resistance against CRKP colonization in antibiotic-perturbed mouse with fecal microbiota transplantation (FMT). Both in vitro and in vivo experiments revealed that Lactiplantibacillus plantarum and Bifidobacterium longum as significant contributors to the decolonization of CRKP. Furthermore, we showed that probiotic supplementation or FMT significantly improved CRKP colonization resistance. The findings highlight that a specific gut microbiome is essential for resisting CRKP colonization, and that targeted microbiome restoration may serve as a viable strategy to prevent CRKP colonization in ICU patients.