Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42-75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MS(E)-based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p ≤ 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT(1A) receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process.