Diatoms are a major phylum of microalgae, playing crucial ecological roles. They derive from secondary endosymbiosis, leading to a complex intracellular architecture. Their ability to store oil in lipid droplets (LDs) upon unfavourable conditions has raised interest for applications, in particular biofuels, yet Lipid Droplet (LD) biogenesis mechanisms in these organisms remain poorly understood. Here, we functionally characterize the homolog of Seipin, a major actor of LD biogenesis, in Phaeodactylum tricornutum. We used an in silico approach to analyze the evolutionary origin of PtSeipin and its specific features. Then, we used a functional genetics approach with a combination of confocal and electronic microscopy and lipidomics to characterize the protein function. We provide evidence that Stramenopiles Seipins were inherited from the host during secondary endosymbiosis. The localization of PtSeipin highlights participation of the plastid's most external membrane in LD biogenesis. Finally, the knock-out (KO) of PtSeipin leads to a strong increase of triacylglycerol (TAG) accumulation, a feature that had not been observed in adipogenic or oleaginous cells and is greatly enhanced following high light exposure. Our results suggest a redirection of lipid fluxes toward TAG synthesis, reduced TAG recycling or a combination of both in PtSeipin KO.