Obesity and vitamin D deficiency are two major public health concerns. Evidence suggests that alteration in gut microbiota composition is a possible risk factor for obesity. Additionally, altered vitamin D status has a potential role in shaping the gut microbial community. Further, the prevalence of obesity has been rising in the Middle East, especially among women of reproductive age, which is of specific concern due to its adverse effects on the health of their offspring. To date, limited evidence is available on the association between gut microbiota composition and vitamin D levels in Arab women. This study aims to identify the associations between serum vitamin D, gut microbiota, and obesity among Saudi females. The current study is a case–control study including 92 women aged 18 to 25 years, (n = 48) with normal weight and (n = 44) with obesity. Anthropometric, biochemical, lifestyle data, and fecal samples were collected and analyzed. We used shotgun metagenomic sequencing to characterize microbial communities of stool samples. Vitamin D levels were significantly associated with alpha and beta diversities. Serum vitamin D levels were positively associated with bacteria known to regulate immunological responses; Bacteroides thetaiotaomicron in the normal weight group (r = 0.34, p = 0.03) and Bifidobacterium adolescentis in the obesity group (r = 0.33, p = 0.04). In conclusion, the findings suggest that vitamin D status may play a role in regulating the gut microbiota composition by inhibiting the growth of pathogenic bacteria while nourishing the beneficial strains.