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Objective: To investigate the relationship between extracellular signal-regulated kinase (ERK) pathway, multidrug resistance gene (mdr-1), ribonucleotide reductase M1 (RRM1) gene and their roles in gemcitabine (GEM) chemoresistance in pancreatic cancer cell line SW1990. Methods: The GEM-resistance cell model was constructed by a stepwise method. Immunohistochemistry was used to measure the expression of ERK protein (ERK1/2) in the established cell strains in a semiquantitative way. The mRNA expression of mdr-1 and RRM1 were detected by RT-PCR. MTT assay was performed to determine the IC50 value. Results: The established GEM-resistant cell strains were able to gain stable growth and passage ability in the medium contained different concentration levels of GEM (0, 30, 60, 100, 150 and 200 nmol/L). The expression of ERK protein, mdr-1 and RRM1 gene were elevated accompanied by the increase of GEM concentration. There was a highly positive correlation between mdr-1, RRM1 expression and GEM-resistance level (r = 0.960, P = 0.002 and r = 0.966, P = 0.002). The expression of ERK protein also correlated with the mdr-1 and RRM1 level (r = -0.943, P = 0.005 and r = -0.883, P = 0.02). At the GEM-resistance level of 200 nmol/L, the grey scale value of ERK1/2 was 164.22 ± 13.17, mdr-1/βactin and RRM1/β-actin were 1.41 ± 0.04 and 1.45 ± 0.18, respectively; after treated with ERK pathway inhibitor U0126, these values synchronously decreased to 186.85 ± 13.14, 0.2 3 ± 0.02 and 0.21 ± 0.03, respectively; inversely, the ERK1/2 grey scale value was 106.55 ± 16.45, mdr-1/βactin and RRM1/β-actin were 1.50 ± 0.07 and 1.52 ± 0.12, respectively, which presented a tendency of synchronously increase after treated with ERK pathway activator EGF. The IC50 values in GEM-resistant cells of 0 nmol/L and 200 nmol/L levels were 4.104 and 10.20, respectively. After treated with U0126, these values decreased to 3.26 and 4.50, respectively; while treated with EGF, the IC50 values increased to 8.89 and 17.17, respectively. Conclusion: The ERK pathway may induce the GEM-chemoresistance in pancreatic cell line SW1990 through the participation in the regulation of the mdr-1 and RRM1 gene expression.