Vitamin D supplementation studies in various pleiotropic outcomes often yield conflicting results. This complexity arises from various factors, including individual differences (baseline Vitamin D levels, genetics, ethnicity, age, and gender). This review aims to clarify the complexities in Vitamin D supplementation research by examining various influencing factors, ultimately providing a comprehensive understanding to guide future studies and offer more accurate insights into the health impacts of Vitamin D supplementation. For this review, we searched PubMed, Google Scholar, and ScienceDirect, analyzing observational studies, meta-analyses, and randomized controlled trials to identify key factors influencing the efficacy of Vitamin D supplementation. By synthesizing findings from diverse research, we aimed to illuminate the nuances shaping the outcomes of these trials. In conclusion, the review suggests that several demographic and biological factors such as baseline 25(OH)D levels, age, ethnicity, genetics, body mass index, diet, sun exposure, medications, comorbid conditions, socioeconomic status, and self-supplementation all play significant roles in the outcomes of Vitamin D supplementation trials. In addition, the dose and duration of therapy, choice of daily versus bolus dosing, route of administration, and the role of free and bound forms of Vitamin D contribute to the complexity of trial results. Vitamin D's pleiotropic effects extend beyond calcium regulation, impacting various health aspects. Inadequate blood levels can confound trial outcomes, emphasizing the importance of reaching appropriate 25(OH)D thresholds. Study design, sample size, bias minimization, and methodology are critical in influencing trial outcomes and designing studies that account for baseline levels and compliance is crucial for meaningful and accurate results. Standardized assays and internationally agreed-upon cutoff levels are essential to mitigate variability in 25(OH)D measurements and improve result reliability.