This study aimed to evaluate if single-nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) gene are associated with gene expression in human periodontal ligament (hPDL) fibroblasts under simulated orthodontic compressive force. hPDL samples from 57 patients were used. A physiological compressive strain was performed to simulate orthodontic tooth movement in pressure areas under cell culture conditions. The RNA from hPDL fibroblasts was isolated to determine the relative gene expression (mRNA) of the VDR. The DNA was also isolated for the genotyping analysis of five SNPs in the VDR gene: BglI (rs739837, G/T), BsmI (rs1544410, T/C), ApaI (rs7975232, A/C), FokI (rs2228570, A/G), and TaqI (rs731236, A/G). Real-time polymerase chain reaction was used for both analyses. Kruskal-Wallis tests were used to compare VDR expression among genotypes of each SNP. A linear regression analysis was performed to evaluate SNP-SNP interaction. An established alpha of 5% was used. The relative mRNA VDR expression according to the genotypes in the SNPs BglI, BsmI, ApaI, FokI, and TaqI was not statistically significantly different (p > 0.05). The SNP-SNP interaction evaluated by regression analysis did not demonstrate any statistically significant association. No association was observed (p > 0.05). In conclusion, the SNPs BglI (rs739837), BsmI (rs1544410), ApaI (rs7975232), FokI (rs2228570), and TaqI (rs731236) did not show an impact on VDR gene expression in hPDL fibroblasts under simulated orthodontic compressive force.