Background Some studies revealed that despite having sufficient sun exposure and dietary supply, the level of serum 25(OH)D in Bangladeshi adults is lower than its normal range. Genetic pattern of an individual is also an essential factor that regulates the level of serum 25(OH)D. However, the genetic variations of CYP2R1 (rs10741657) and their association with low serum 25(OH)D level in Bangladeshi adults are yet to be explored. Objective This study was conducted to determine the frequency of variants of rs10741657 of CYP2R1 gene and its association with low serum 25(OH)D level among Bangladeshi adults. Method This pilot study was conducted among thirty individuals with low serum 25(OH)D level as the study population and ten subjects with sufficient serum 25(OH)D level as controls based on the inclusion and exclusion criteria. Genetic analysis of rs10741657 of CYP2R1 including primer designing, DNA extraction, PCR of target region with purification and Sanger sequencing of the PCR products were done accordingly. For statistical analysis, One-way ANOVA followed by LSD test, Freeman-Halton extension of Fisher’s exact test, Chi-square test (χ2) test and unpaired student t-test were performed. Results In this study, genetic variants of CYP2R1 (rs10741657) among the study population were genotype GG (63.30%), GA (30%) and AA (6.7%). Minor allele frequency of the study population was 0.217. The association between GG and GA genotypes of CYP2R1 (rs10741657) with low serum 25(OH)D level among the study population was found and it was statistically significant. Statistically significant differences were also observed between the genotypes and alleles of the study population and controls. Conclusions The presence of ‘GG’ and ‘GA’ genotypes of rs1041657 in CYP2R1 gene is associated with low serum 25(OH)D level among Bangladeshi adults in this pilot study.