Objective To silence polβ gene by RNAi technique and to observe its effects on the drug-resistance of esophageal carcinoma cells to cisplatin (cDDP). Methods We constructed recombinant lentivirus containing siRNA targeting polβ genes. The two lentiviral vectors containing siRNA targeting the polβ gene were designated as pRNAT-U6.2/Lenti-polβ1 and pRNAT-U6.2/Lenti-polβ2, and the negative control vector as pRNAT-U6.2/Lenti-polβC; they were used to infect EC9706/cDDP cells, which was resistant to cDDP. The expression of polβ was then examined by RT-PCR, Western blotting and immunofluorescence. MTT was used to observe the inhibitory effect of cDDP; the IC₅₀ of cDDP and resistance index (RI) were calculated. Results Both pRNAT-U6.2/Lenti-polβ1 and pRNAT-U6.2/Lenti-polβ2 down-regulated polβ at mRNA and protein levels, with the effect of former being more prominent. Cisplatin inhibited EC9706/cDDP cell proliferation in a dose-dependent manner. The IC₅₀ values of cDDP in cells infected with pRNAT-U6.2/Lenti-polβ1, pRNAT-U6.2/Lenti-polβC and untreated EC9706/cDDP were 55.71 μg/mL, 62.41 μg/mL, and 63.11 μg/mL, respectively, with the first one being significantly different from the latter two (P<0.05); and the corresponding RI values were 13.9, 15.5, and 15.7, respectively, also with the first one being significantly different from the latter two(P<0.05). Results It is suggested that polβ expression is associated with cDDP resistance in EC9706/cDDP, and silencing polβ by RNAi can partly reverse the drug resistance of EC9706/cDDP to cDDP.