AbstractTocopherols, collectively known as vitamin E, are lipophilic antioxidants, essential dietary components for mammals and exclusively synthesized by photosynthetic organisms. Of the four forms (α, β, γ and δ), α‐tocopherol is the major vitamin E form present in green plant tissues, and has the highest vitamin E activity. Synthetic α‐tocopherol, being a racemic mixture of eight different stereoisomers, always results less effective than the natural form (R,R,R) α‐tocopherol. This raises interest in obtaining this molecule from natural sources, such as plant cell cultures. Plant cell and tissue cultures are able to produce and accumulate valuable metabolites that can be used as food additives, nutraceuticals and pharmaceuticals. Sunflower cell cultures, growing under heterotrophic conditions, were exploited to establish a suitable in vitro production system of natural α‐tocopherol. Optimization of culture conditions, precursor feeding and elicitor application were used to improve the tocopherol yields of these cultures. Furthermore, these cell cultures were useful to investigate the relationship between α‐tocopherol biosynthesis and photomixotrophic culture conditions, revealing the possibility to enhance tocopherol production by favouring sunflower cell photosynthetic properties. The modulation of α‐tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.