Background: Iron deficiency is the most widespread nutritional deficiency worldwide, and it is the primary cause of anemia, particularly in low- and middle-income countries such as Indonesia. Iron deficiency has a multifactorial etiology involving complex interactions between genetic factors, especially the transmembrane protease serine 6 (TMPRSS6) rs855791 variant, which encodes matriptase-2, a protein involved in regulating hepcidin expression, and nongenetic factors, including sociodemographics, nutritional status, iron intake, and menstrual patterns. Women of reproductive age, including medical students, are susceptible to iron deficiency owing to unhealthy dietary habits, growth requirements, and menstruation. Iron deficiency among medical students may lead to decreased academic performance and productivity. Additionally, as future mothers, women may experience a heightened risk of delivering children with intellectual disabilities and various psychosocial impairments. Owing to the significant consequences of this condition, identifying the underlying causes of iron deficiency is crucial. The high prevalence of iron deficiency in Indonesia poses a challenge in addressing these contributing factors to effectively reduce its occurrence. Objective: This study aims to investigate the association of the TMPRSS6 rs855791 variant and nongenetic factors with iron deficiency among female medical students in Yogyakarta, Indonesia. Methods: This is a case-control study. We will recruit female medical students from the Faculty of Medicine, Public Health, and Nursing of Universitas Gadjah Mada in Yogyakarta, Indonesia. The inclusion criteria are being a final-year female undergraduate medical student who has not entered the clinical clerkship phase, aged 18 to 24 years, not pregnant, providing written consent, and having no history of chronic and inflammatory diseases, congenital diseases, hematological disorders, or blood transfusions during the last 3 months. Participants will be excluded if the C-reactive protein level is higher than 5 mg/L. Participants will be further grouped according to iron status criteria. Profiles of hemogram and iron markers will be compared between the case and control groups using the independent samples 2-tailed t test or the Mann-Whitney U test, while genotype and allele frequencies will be analyzed using the chi-square test. One-way ANOVA or the Kruskal-Wallis test will be used to assess the impact of different genotypes on iron marker levels. Multivariate analysis will be performed with logistic regression to determine factors independently associated with iron deficiency risk. P≤.05 will be considered statistically significant. Results: The study received funding in January 2025. Data collection began in February 2025 and is anticipated to conclude by October 2025. At the time of manuscript submission, 115 participants had been enrolled. The study findings are expected to be published in 2026. Conclusions: This study will determine the interaction between the TMPRSS6 rs855791 variant and nongenetic factors that contribute to the risk of iron deficiency among female medical students in Yogyakarta, Indonesia.