Cystic fibrosis (CF) patients and CFTR knock-out mice (CF mice) display an imbalance in fatty acids with high arachidonic acid (AA) and low docosahexaenoic acid (DHA) concentrations. Our recent studies demonstrated defects in another class of lipids, ceramides, in CF patients and CF mice. This study investigates the relationship between ceramide, AA, DHA and the correction of lipid imbalances in CF mice following treatment with fenretinide. Concentrations of AA, DHA and ceramide were assessed in plasma from 58 adult CF patients and 72 healthy controls. Following 28-day treatment with fenretinide, the same analysis was preformed in wild-type and CF mice from plasma and organs (lung, ileum, pancreas and liver). Low ceramide levels were associated with high AA and low DHA concentrations in CF patients. No correlation was observed in healthy controls. Greater deficiencies were seen in CF patients who were diagnosed before the age of 18, specifically with statistically significant higher levels of AA.Treatment with fenretinide (N-(4-hydroxyphenyl)retinamide; 4-HPR) normalized high levels of AA, low levels of ceramide and increased the levels of DHA in CF mice. As in CF patients, low ceramide levels correlated with higher AA and lower DHA levels in plasma of CF mice. Lipid abnormalities correlated with ceramide deficiencies in CF patients and CF mice. We found that fenretinide treatment normalizes the fatty acid imbalance in CF mice with reducing AA to WT levels and increasing DHA. We propose that fenretinide treatment might improve this pathological phenotype in CF patients.