[Objective]　The　aim　of　the　study　is　to　explore　the　feasibility　of　ion　exchange　chromatography　as　a　pretreatment　method　for　rat　fecal　samples.　[Method]　Rat　fecal　samples　were　collected　and　pretreated　with　three　different　methods:　control　(Con)　group,　centrifugation　(Cen)　group,　and　ion　exchange　chromatography　(Chr)　group.　The　DNA　extraction　quality,　16S　rRNA　amplicon　high-throughput　sequencing　results,　and　real-time　fluorescence　quantitative　PCR　detection　results　of　specific　bacterial　groups　were　compared　between　samples　pretreated　with　different　methods.　[Result]　The　mass　concentration　of　DNA　extracted　from　samples　of　the　Chr　group　was　1.44　times　that　of　the　Cen　group　and　6.49　times　that　of　the　Con　group.　The　number　of　operational　taxonomic　unites　(OTUs)　in　the　Con　group　was　2　630,　while　the　number　increased　to　3　220　and　3　223　in　the　Cen　group　and　Chr　group,　respectively.　α　diversity　and　β　diversity　analyses　indicated　that　the　Chr　group　had　higher　microbial　diversity,　higher　similarity　and　repeatability　among　the　samples　within　the　group.　The　results　of　real-time　fluorescence　quantitative　PCR　showed　that　the　abundance　of　Bifidobacterium　adolescentis　was　different　among　the　three　groups.　Specifically,　the　abundance　of　Bifidobacterium　adolescentis　was　the　highest　in　the　Chr　group,　moderate　in　the　Cen　group,　and　the　lowest　in　the　Con　group.　When　the　volume　of　bacterial　suspension　added　increased　from　100　μL　to　200　μL,　there　was　no　significant　difference　in　the　abundance　of　Bifidobacterium　adolescentis　in　the　Con　group　(P＞0.05),　while　the　abundance　increased　to　1.32　times　in　the　Cen　group　and　1.70　times　in　the　Chr　group.　This　indicated　that　ion　exchange　chromatography　better　preserved　the　microorganisms　in　rat　fecal　samples.　[Conclusion]　As　a　pretreatment　method　of　rat　fecal　samples,　ion　exchange　chromatography　can　improve　the　repeatability　and　sensitivity　of　the　analysis　of　intestinal　flora.　©　2025　Editorial　office　of　Journal　of　Food　Science　and　Biotechnology.　All　rights　reserved.