Nutrition knowledge is necessary for individuals to adopt appropriate dietary habits, and needs to be evaluated before nutrition education is provided. However, there is no tool to assess general nutrition knowledge of adults in Japan. Our aims were to determine the validity and reliability of a general nutrition knowledge questionnaire for Japanese adults. We developed the pilot version of the Japanese general nutrition knowledge questionnaire (JGNKQ) and administered the pilot study to assess content validity and internal reliability to 1,182 Japanese adults aged 18-64 y. The JGNKQ was further modified based on the pilot study and the final version consisted of 5 sections and 147 items. The JGNKQ was administered to female undergraduate Japanese students in their senior year twice in 2015 to assess construct validity and test-retest reliability. Ninety-six students majoring in nutrition and 44 students in other majors who studied at the same university completed the first questionnaire. Seventy-five students completed the questionnaire twice. The responses from the first questionnaire and both questionnaires were used to assess construct validity and test-retest reliability, respectively. The students in nutrition major had significantly higher scores than the students in other majors on all sections of the questionnaire ( p =0.000); therefore, the questionnaire had good construct validity. The test-retest reliability correlation coefficient value of overall and each section except “The use of dietary information to make dietary choices” were 0.75, 0.67, 0.67, 0.68 and 0.61, respectively. We suggest that the JGNKQ is an effective tool to assess the nutrition knowledge level of Japanese adults.