Background: The etiopathogenesis of vernal keratoconjunctivitis (VKC), is incompletely understood. Bioactive lipids play a key role in the allergic disorders and there are no studies exploring it in the ocular surface allergic disorder such as VKC. This study focused on the sphingolipid metabolism in VKC and explored the association of ocular surface sphingolipids with the refractory nature of the disease. Methods: Active VKC cases and age-matched healthy controls were recruited as part of a one-year prospective study at our tertiary eye care centre in South India. Imprint cytology was used to assess gene expression of enzymes of sphingolipids metabolism in conjunctival cells. Sphingolipids levels were estimated in the tears by LC-MS/MS analysis. Systemic levels of Sphingosine-1-phosphate (S1P) and Ceramide-1-Phosphate and IgE were estimated by ELISA. IgE induced regulation of S1P receptor gene expression was assessed by western blot analysis of histone deacetylases in cultured mast cells. Results: Significantly altered gene expression of the sphingolipids enzymes was noted in VKC in conjunctival cells. Pooled tears showed significantly lowered levels of S1P (d17:1), S1P (d17:0) and S1P (d20:1) in VKC. Additionally, Cer (d18:/17:0) was significantly lowered in R-VKC compared to NR-VKC. Lowered C1P and raised IgE were observed at serum level in VKC. Increased S1P receptors and histone deacetylases expression indicate regulation by histone modification as evaluated in IgE induced mast cells. Conclusion: Altered sphingolipid metabolism is associated with VKC pathogenesis and is differential in refractory cases that showed lower ceramide and specific sphingosines compared to non-refractory VKC.