Cystic fibrosis (CF) is an autosomal recessive disorder, caused by genetic mutations in CF transmembrane conductance regulator protein. Several reports have indicated the presence of specific fatty acid alterations in CF patients, most notably decreased levels of plasmatic and tissue docosahexaenoic acid (DHA), the precursor of specialized pro-resolving mediators. We hypothesized that DHA supplementation could restore the production of DHA-derived products and possibly contribute to a better control of the chronic pulmonary inflammation observed in CF subjects. Sputum samples from 15 CF and 10 chronic obstructive pulmonary disease (COPD) subjects were collected and analyzed by LC/MS/MS, and blood fatty acid were profiled by gas chromatography upon lipid extraction and transmethylation. Interestingly, CF subjects showed increased concentrations of leukotriene B4 (LTB4), prostaglandin E2 (PGE2), and 15-hydroxyeicosatetraenoic acid (15-HETE), when compared with COPD patients, whereas the concentrations of DHA metabolites did not differ between the two groups. After DHA supplementation, not only DHA/arachidonic acid (AA) ratio and highly unsaturated fatty acid index were significantly increased in the subjects completing the study (p < 0.05) but also a reduction in LTB4 and 15-HETE was observed, together with a tendency for a decrease in PGE2, and an increase in 17-hydroxy-docosahexaenoic acid (17OH-DHA) levels. At the end of the washout period, LTB4, PGE2, 15-HETE, and 17OH-DHA showed a trend to return to baseline values. In addition, 15-HETE/17OH-DHA ratio in the same sample significantly decreased after DHA supplementation (p < 0.01) when compared with baseline. In conclusion, our results show here that in CF patients, an impairment in fatty acid metabolism, characterized by increased AA-derived metabolites and decreased DHA-derived metabolites, could be partially corrected by DHA supplementation.