The General and Sport Nutrition Knowledge Questionnaire (GeSNK) is an instrument that has been developed and validated to assess the level of nutrition knowledge in adolescents and young adults. The aim of the present study was to validate the GeSNK questionnaire in a group of Spanish adolescents in the framework of a Nutrition Education Programme in Secondary Schools in Andalusia, Spain. This cross-sectional questionnaire validation study was developed in two phases: translation-cultural adaptation and validation. A total of 305 adolescents aged 11 to 17 years, studying from the first to the third year of compulsory secondary education, participated on a voluntary basis. The GeSNK questionnaire consists of 62 items: 29 items for the General Nutrition section and 33 items for the Sports Nutrition section. Cronbach’s alpha coefficient for the complete questionnaire (GeSNK Total) was: 0.934; for the GeSNK General Nutrition section it was 0.918; and for the GeSNK Sports Nutrition section it was 0.856. The stability measured by the correlation coefficient for the General Nutrition section was 0.406 (p = 0.000); for the Sports Nutrition section it was 0.198 (p = 0.017); and for GeSNK Total the stability was 0.545 (p = 0.000). The questionnaire also demonstrated adequate construct validity. We therefore conclude that the Spanish version of the GeSNK questionnaire is a valid instrument to measure the level of knowledge in general nutrition and sports nutrition in adolescents.