Objective:To study the relationship of folic acid, homocysteine with pancreatic cancer. Methods: A case-control study was performed in which the case group was defined as patients with newly diagnosed pancreatic cancer and the control group were population-based healthy individuals. ELISA assay was used to determine the plasma levels of folic acid, homocysteine, vitamine B6 and vitamine B12 in all the subjects. The general information (such as demology data, smoking history, diet, etc) was collected by face to face talking using a standard questionnaire. Univariate analyses were performed using Chi-square tests for norminal variables and unpair t-tests for continuous variables. The variables with a P value no more than 0.25 in univariate analyses were selected as candidate variables for a multivariate logistic regression analysis. Results: Forty-two patients with pancreatic cancer and 42 healthy individuals were included in the present study. The results of univariate analyses showed that the plasma folic acid, homocysteine, vitamine B12 and vitamine B6 were not significantly different between the two groups (P＞0.05); they were potentially associated with pancreatic cancer (P<0.25), but were not significant risk factors(P＞0.05).The consumptions of vegetables, fruits, white meat and milk in case group was significantly less in the pancreatic cancer group than in the control group (P<0.05). Multivariate analysis showed that the odds ratios (95% CI) of plasma folic acid, vitamin B6, and homocysteine for pancreatic cancer were 0.571 (0.383-0.851), 0.750 (0.557-1.011), and 1.514 (0.986-2.326), respectively. Conclusion: Increased plasma folic acid can decrease the risk of pancreatic cancer. Plasma vitamin B6 might be a protective factor and homocysteine might be a risk factor of pancreatic cancer.