Objective To investigate the effects of toll-like receptor 9 (TLR9) agonist CpG ODN2216 on the sensitivity of pancreatic cancer cell line PANC1's to gemcitabine.Methods The immunofluorescence staining method and Western blot method were used to examine the expression of TLR9 protein in PANC1 cells.The changes of sensitivity to gemcitabine after CpG ODN2216 treatment were examined by MTT assay.Results The TLR9 protein was highly expressed in PANC1 cells and the median inhibition concentration of gemcitabine against PANC1 cells was reduced from (1.23 ± 0.14) mg/L to (0.28 ± 0.13) mg/L after CpG ODN2216 treatment,and the difference between the two groups was statistically significant (P ＜0.01).After 0.01,0.10,1.00,10.00 mg/L gemcitabine treatment with CpG 0DN2216,the inhibition rates of PANC1 were (34.4 ±1.3)％,(43.5 ± 2.7)％,(76.3 ± 2.5)％,(95.3 ± 2.2)％ ; and without CpG ODN2216,the inhibition rates of PANC1 were (14.5 ± 0.9) ％,(23.5 ± 1.1) ％,(44.8 ± 1.4) ％,(63.6 ± 1.8) ％,and the difference between the two groups was statistically significant (P ＜ 0.01).Conclusions The sensitivity of PANC1 cells to gemcitabine can be enhanced by CpG ODN2216.