Cisplatin (CDDP) is one of the front-line chemotherapeutic drugs used in the treatment of esophageal squamous cell carcinoma (ESCC). Occurrence of resistance to CDDP has become one of the main challenges in cancer therapy. In this study, the gene expression profile of CDDP-resistant ESCC cells was investigated and molecular approaches were explored in an attempt to reverse the CDDP resistance. A CDDP-resistant SLMT-1/CDDP1R cell line was established from SLMT-1 cells by subculturing in the medium containing an increasing concentration of CDDP (0.1⁻1μg/mL). Mitochondrial (MTS) cytotoxicity assay, cell proliferation assay and cell morphology were used to assess the acquisition of cisplatin-resistance. The most differentially expressed gene in SLMT-1/CDDP1R cells was identified by cDNA microarray analysis compared with the parental SLMT-1 cells and validated by quantitative real-time polymerase chain reaction (qPCR). Association between expression of the most differentially expressed target gene to cisplatin-resistance was verified by RNA interference. An attempt to reversecisplatin-resistance phenotypes was made by using the vector expressing the most downregulated target gene in the CDDP-resistant cells. A CDDP-resistant ESCC cell line, SLMT-1/CDDP1R, was established with 2.8-fold increase CDDP-resistance (MTS50 = 25.8 μg/mL) compared with the parental SLMT-1 cells. cDNA microarray analysis revealed that IGFBP5 showed the highest level of downregulation in SLMT-1/CDDP1R cells compared with the parental SLMT-1 cells. Suppression of IGFBP5 mediated by IGFBP5-targeting siRNA in parental SLMT-1 cells confirmed that IGFBP5 suppression in ESCC cells would induce CDDP-resistance. More importantly, upregulation of IGFBP5 using IGFBP5 expression vector reduced cisplatin-resistance in SLMT-1/CDDP1R cells by 41%. Thus, our results demonstrated that IGFBP5 suppression is one of the mechanisms for the acquisition of cisplatin-resistance in ESCC cells. Cisplatin-resistance phenotype can be reversed by increasing the expression level of IGFBP5. The overall findings of this study thus offered a new direction for reversing the CDDP resistance in ESCC and possibly in other cancer types with further investigations in future.