Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare immune disease, accompanied by high mortality. Vitamin D (VD), as a marker of inflammation, plays an important role in the regulation of the immune system by binding to receptors of immune cells (T and B cells). The purpose of this study was to investigate the expression level of vitamin D in HLH and to clarify its relationship with prognosis. Method: We conducted a retrospective analysis of 54 adult sHLH cases from November 2015 to December 2020. Results: Among the cohort, 49 (90.7%) patients with sHLH had VD insufficiency (25-hydroxy vitamin D [25-(OH)D] <50nmol/L) and median level was 23.95 (range: 7.5-90.8) nmol/L. Classified by 30nmol/L as the threshold value, there were no significant differences on baseline characteristics between two groups, except triglycerides (TG). Level of 25-(OH)D was significantly associated with TG (r=-0.34, P=0.011) and ferritin (r=-0.26, P=0.055). Patients with lower 25-(OH)-D level acquired shorter overall survival (28 vs. 125 days P=0.041). After multivariable adjustment, 25-(OH)-D was identified as an independent factor of survival (HR 0.364;95% CI 0.183-0.724; P=0.004). A significant interaction was discovered at 25-(OH)-D with ferritin (P=0.009) through subgroup analysis. Vitamin D is more likely to predict survival in elderly, male, MHLH, EBV infection, hemoglobin<90g/L, platelet<100×109/L, fibrinogen<1.5g/L, triglyceride, ferritin >10000ug/l, sCD25>20000ng/l group. Conclusions: Patients with sHLH almost had vitamin D insufficiency and vitamin D is an important prognostic protective factor for sHLH.