Abstract: Background and Objective: Many polymorphisms had been mapped in vitamin D receptor (VDR) gene on chromosome 12, they had variable location which influences its functional prospect. The present study aimed to evaluate the possible association between VDR gene Fok I SNP (C>T, rs2228570) polymorphism and Spontaneous Preterm Birth (SPTB) in the Egyptian pregnant population. Materials and Methods: A total of 160 pregnant women, divided into two groups: 80 preterm (case group) and 80 control groups, were investigated for VDR gene Fok I polymorphism by predesigned Taq-Man SNPs genotyping assay. Results: The Odds Ratio (OR) for the preterm birth risk was significantly higher with the mutant homozygous (TT) genotype (p = 0.01). Vitamin D level is significantly different among the three VDR Fok I SNP genotypes in both groups. The lowest vitamin D level was found in variant homozygous TT genotype (19.6 and 25.5 ng mL–1 in preterm and control groups, respectively). The C allele was associated with low preterm birth risk [OR: 0.43 (0.20-0.91)] and the T allele was associated with high preterm labor risk [OR: 2.0 (1.04-3.79)]. Conclusion: The VDR Fok I polymorphism is associated with an increased risk of Spontaneous Preterm Birth (SPTB) in Egyptian pregnant women. It may play a possible role in SPTB etiology. The current study provides data on the consideration of VDR Fok I polymorphism as a potential biomarker for SPTB.