Background:　Chemotherapy　and　immunotherapy　have　improved　the　cure　rate　and　survival　period　for　colorectal　cancer　(CRC),　but　genetic　heterogeneity　among　patients　leads　to　chemotherapy　resistance　and　disease　progression.　Identifying　new　molecular　markers　is　crucial　for　improving　prognosis　for　CRC　patients.　KIR2DL4,　a　transmembrane　glycoprotein　expressed　by　immune　cells,　has　shown　potential　therapeutic　and　prognostic　value　in　other　cancers,　but　in　CRC　remains　unclear.　Methods:　This　study　validated　the　expression　levels　of　KIR2DL4　in　CRC　by　integrating　multiple　public　databases　and　assessed　through　immunohistochemistry　(IHC).　We　further　evaluated　the　diagnostic　and　prognostic　value　of　KIR2DL4　and　explored　correlation　with　immune　cell　infiltration　and　chemotherapy　sensitivity.　The　role　of　KIR2DL4　was　further　validated　through　functional　enrichment　analysis.　Cellular　assays　were　conducted　using　CCK8,　colony-formation　assay　and　scratch　wound　assay.　Results:　The　study　found　that　KIR2DL4　is　significantly　downregulated　in　CRC　and　closely　associated　with　poor　prognosis.　The　low　expression　of　KIR2DL4　is　associated　with　decreased　immune　cell　infiltration　and　reduced　chemotherapy　sensitivity.　Functional　enrichment　analysis　suggests　that　KIR2DL4　may　inhibit　development　of　CRC　by　affecting　immune　cell　infiltration　and　modulating　chemotherapy　sensitivity.　Cellular　assays　have　confirmed　that　inhibiting　KIR2DL4　significantly　promotes　the　proliferation　and　migration　of　CRC.　Inhibition　of　KIR2DL4　expression　significantly　decreased　the　chemosensitivity　of　CRC　cells　to　oxaliplatin　and　5-FU.　Conclusion:　The　significant　downregulation　of　KIR2DL4　in　CRC,　associated　with　CRC　metastasis　and　poor　prognosis,　highlights　its　importance　as　a　potential　new　biomarker　for　treatment　and　prognosis　assessment　of　CRC.　Future　research　should　delve　into　the　molecular　mechanisms　of　KIR2DL4　and　potential　applications　in　regulating　immunotherapy　and　chemotherapy　sensitivity.　©　2024　The　Authors