Iron (Fe) is an essential micronutrient, and deficiency in available Fe is one of the most important limiting factors for plant growth. In some species including Medicago truncatula, Fe deficiency results in accumulation of riboflavin, a response associated with Fe acquisition. However, how the plant's Fe status is integrated to tune riboflavin biosynthesis and how riboflavin levels affect Fe acquisition and utilization remains largely unexplored. We report that protein kinase CIPK12 regulates ferric reduction by accumulation of riboflavin and its derivatives in roots of M. truncatula via physiological and molecular characterization of its mutants and over-expressing materials. Mutations in CIPK12 enhance Fe accumulation and improve photosynthetic efficiency, whereas overexpression of CIPK12 shows the opposite phenotypes. The Calcineurin B-like proteins CBL3 and CBL8 interact with CIPK12, which negatively regulates the expression of genes encoding key enzymes in the riboflavin biosynthesis pathway. CIPK12 negatively regulates Fe acquisition by suppressing accumulation of riboflavin and its derivatives in roots, which in turn influences ferric reduction activity by riboflavin-dependent electron transport under Fe deficiency. Our findings uncover a new regulatory mechanism by which CIPK12 regulates riboflavin biosynthesis and Fe-deficiency responses in plants.