Objective: To construct the plasmid expression vector that expresses short hairpin RNA (shRNA) against CXC chemokine receptor 1(CXCR1). Methods: Three shRNA expression vectors targeting CXCR1 gene and one negative control (non-targeting sequence) expression vector were designed and constructed according to the mRNA sequence of CXCR1 gene and RNA interference design guidelines. After identification with restriction enzyme digestion and sequencing analysis, these vectors were transfected into gastric cancer MKN45 cells in vitro, and then the CXCR1 mRNA and protein expressions in the cells were detected by RT-PCR and Western blot analysis. Results: As identified by enzyme digestion and sequencing analysis, all the three shRNA eukaryotic expression plasmid vectors targeting CXCR1 gene were successfully constructed. Compared with untransfected MKN45 cells or MKN45 cells transfected with negative control vector, both mRNA and protein levels of CXCR1 were significantly reduced in MKN45 cells transfected with any of the three shRNA vectors (all P<0.05). Conclusion: The successful construction of plasmid expression vector encoding shRNA against CXCR1 may provide a preliminary step for further investigation of the role of CXCR1 in gastric cancer and experimental targeted therapy.