The hypostasis of the One is a fundamental concept in Plotinian metaphysics, and its relationship to Presocratic philosophy raises an important question in the history of Greek thought. This study examines how different Presocratic conceptions of unity were critically reconfigured in Plotinus’ formulation of the One through the conceptual mediation of Plato. Employing an analytical-interpretive method and a comparative approach, it draws on Plotinus’ writings alongside accounts and interpretations of Presocratic philosophy. The analysis considers Parmenides, the Pythagoreans, Heraclitus, Empedocles, and Anaxagoras. It shows that earlier accounts generally conceived unity in relation to being, cosmic order, multiplicity, or intellect, while the Pythagorean monad offered conceptual possibilities for a negative account of unity. Parmenidean unified Being is more closely associated with the Plotinian hypostasis of Intellect than with the One. Heraclitean unity and Empedoclean Love function as unifying principles within multiplicity rather than as absolute first principles. Although Anaxagoras’ unmixed Nous is independent of matter and orders the cosmos, its cognitive and determinate character prevents its identification with the One. Plotinus selectively preserves the conceptual possibilities of these traditions while relocating the first principle beyond being. Accordingly, the Plotinian One is neither a direct continuation nor a simple synthesis of Presocratic doctrines, but the outcome of their critical reconfiguration within a Platonic metaphysical framework. This transformation establishes a fundamental distinction between unity as a property or ordering principle of beings and unity as their transcendent source.