Although erlotinib has become an important therapeutic option in addition to gemcitabine, the high frequency of KRAS mutations in pancreatic cancer probably limits the benefits. We retrospectively studied 136 pancreatic cancer patients with available formalin-fixed paraffin-embedded tumor blocks from 2003 to 2009 to understand the clinical significance of KRAS mutations in pancreatic cancer patients treated with gemcitabine-based chemotherapy. KRAS mutations were analyzed by sequencing codons 12, 13, and 61. In this study, 71 (52.2%) of the 136 pancreatic adenocarcinomas examined harbored a point mutation in codons 12 (n = 70) and 61 (n = 1) of KRAS. KRAS mutation was not associated with clinicopathologic parameters. Patients with KRAS mutations showed a worse response (11.3%) than those with wild-type KRAS (26.2%) and poor survival (mutant KRAS, 5.8 months vs. wild-type KRAS, 8.0 months; P = 0.001). Multivariate analysis revealed good prognostic factors for overall survival as well to moderately differentiated histology (P < 0.001; HR = 0.437, 95% CI: 0.301-0.634), locally advanced disease (P < 0.001; HR = 0.417, 95% CI: 0.255-0.681), response to first-line chemotherapy (P = 0.003; HR = 0.482, 95% CI: 0.297-0.780), and wild-type KRAS (P = 0.001; HR = 0.523, 95% CI: 0.355-0.770). However, the observed survival advantage is derived from the subgroup of patients treated with gemcitabine/erlotinib (9.7 vs. 5.2 months; P = 0.002), whereas no survival difference based on KRAS mutation status is obvious in the other subgroup of patients treated without erlotinib (7.0 vs. 7.0 months; P = 0.121). These results need to be further explored in upcoming prospective studies to provide a rationale for personalized medicine in pancreatic cancer.