Introduction The level of expression of the immunoregulatory human leukocyte antigen-G (HLA-G) has been suggested to play a role in the immunopathogenesis of systemic lupus erythematosus (SLE). A 14 bp insertion/deletion (ins/del) polymorphism in the 3ˊuntranslated region of HLA-G gene may influence the level of expression. The role of Toll-like receptor 9 (TLR9) in the pathogenesis of SLE has been highlighted. Data among Egyptian patients are quite limited. Purpose To detect the association of HLA-G 14 bp ins/del gene polymorphism with the susceptibility to SLE and to correlate TLR9 serum level with disease activity among Egyptian patients. Patients and Methods A case-control study that included 102 SLE female patients and 102 healthy matched volunteers as controls was carried out. Disease activity in patients was determined using the modified Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). HLA-G 14 bp ins/del genotype was detected by polymerase chain reaction (PCR). TLR9 serum level was estimated using enzyme-linked immunosorbent assay (ELISA) technique. Results The ins/ins genotype was significantly increased among SLE patients compared to healthy subjects (58.8% vs 9.8%; odds ratio [OR] = 11.79, P < 0.001). The 14 bp ins allele was significantly more frequent in SLE patients than in healthy subjects (65.7% vs 27.9%, respectively) and significantly associated with an increased risk of SLE (OR 4.94, P < 0.001). The mean TLR9 serum level showed a significant increase in SLE patients compared to healthy subjects (397.04±137.86 vs 195.22±45.14 ng/L, p < 0.001) and was significantly associated with disease activity as well as to patients’ HLA-G genotypes (p < 0.001). Conclusion Among Egyptian population, HLA-G 14 bp ins/ins homozygous genotype and ins allele may constitute a potential risk for SLE susceptibility, while TLR9 serum level is significantly associated with disease activity.