Background: Patients with systemic lupus erythematosus (SLE) are prone to develop vitamin D (25(OH) D3) deficiency, due to several factors and there is an association between lower vitamin D levels and higher SLE disease activity. The aim of this research was to assess the prevalence of vitamin D deficiency in Egyptian female patients with SLE. Furthermore, we analyzed the potential relationship between this deficiency and SLE manifestations, disease activity, and its effect on interferon alpha (IFN-a) gene expression and serum level. Methods: We evaluated the serum levels of vitamin D 25(OH)D3 and IFN-a by enzyme-linked immunosorbent assay (ELISA). IFN-a gene expression was measured by real-time polymerase chain reaction (PCR) assay in 123 Egyptian female patients with SLE and in 100 females as a healthy control group. Results: Vitamin D deficiency was prevalent in 20.30%, while insufficiency was prevalent in 42.40% of the total group of patients. Serum levels of 25(OH)D3 were significantly decreased in the group of severe disease, and in the group of patients with lupus nephritis. 25(OH)D3 showed highly significant negative correlation with the SLE Disease Activity Index (SLEDAI) in the high activity group and lupus nephritis group. There was a significant negative correlation between 25(OH)D3 and IFN-a serum level and gene expression in all patients; more significant in the group with lupus nephritis. Conclusions: The deficiency of 25(OH)D3 has a direct relationship with increase disease activity and nephritis in Egyptian SLE patients, suggesting the need for vitamin D supplementation in these patients. Also, it is directly correlated with increased secretion and gene expression of IFNa, suggesting its role in pathogenesis of lupus nephritis, to be confirmed by further longitudinal observational studies. Lupus (2018) 27, 199–209.