The aim of the present study was to examine the relative validity of foods and nutrients calculated by a new food frequency questionnaire (FFQ) in the Norwegian Mother and Child Cohort Study (MoBa). Reference measures were a 4-day weighed food diary (FD), a motion sensor for measuring total energy expenditure, one 24-h urine collection for analysis of nitrogen and iodine excretion, and a venous blood specimen for analysis of plasma 25-hydroxy-vitamin D and serum folate. A total of 119 women participated in the validation study, and 112 completed the motion sensor registration. Overall, the level of agreement between the FFQ and the FD was satisfactory, and significant correlations were found for all major food groups and for all nutrients except vitamin E. The average correlation coefficient between the FFQ and the FD for daily intake was 0.48 for foods and 0.36 for nutrients, and on average, 68% of the participants were classified into the same or adjacent quintiles by the two methods. Estimated total energy expenditure indicated that under-reporting of energy intake was more extensive with the FD than with the FFQ. The biological markers confirmed that the FFQ was able to distinguish between high and low intakes of nutrients, as measured by vitamin D, folate, protein and iodine. This validation study indicates that the MoBa FFQ produces reasonable valid intake estimates and is a valid tool to rank pregnant women according to low and high intakes of energy, nutrients and foods.