Biotin, also known as vitamin H or B8, is an essential cofactor for CO2-manipulating enzymes found in all three domains of life. The past few years have seen decisive progress accomplishments on the elucidation of biotin metabolism in plants, at both the molecular and cellular levels, and several unique features are emerging. Noticeably, biotin synthesis in plants is split between cytosol and mitochondria. Biotin-utilizing enzymes are also quartered between different compartments of the plant cell. Among these compartments, mitochondria play a central role. In this review, I will summarize the most recent discoveries about the synthesis, manipulation and compartmentalization of biotin in plant cells. These advances open challenging prospects for plant biotechnology purposes through a better understanding of regulation, storage and utilization of the vitamin. Understanding how the biotin biosynthetic pathway interacts with other metabolic pathways and the emerging involvement of mitochondria in plant growth and development, through its intimate implication in vitamins synthesis are also particularly challenging.