There is a need to account for the content of 25-hydroxyvitamin D3 (25OH-D3) in foods to more accurately estimate dietary vitamin D intake, given its higher biological activity. A high-performance liquid chromatography–tandem mass spectrometry method was applied to the determination of vitamin D3 and 25OH-D3 in bovine milk obtained during early lactation and over the course of a full milking season. In this seasonal study of bovine milk, vitamin D3 levels ranged from 167 ng L−1 in winter to 615 ng L−1 in summer, whereas the content of 25OH-D3 in bovine milk was <50 ng L−1 and showed little variation. This study will provide manufacturers with data concerning endogenous vitamin D content that will enhance formulation capability related to the production of bovine-milk-based paediatric products.