The unicellular marine diatom Phaeodactylum tricornutum accumulates up to 35% eicosapentaenoic acid (EPA, 20:5n3) and has been used as a model organism to study long chain polyunsaturated fatty acids (LC-PUFA) biosynthesis due to an excellent annotated genome sequence and established transformation system. In P. tricornutum, the majority of EPA accumulates in polar lipids, especially in galactolipids such as mono- and di-galactosyldiacylglycerol (MGDG and DGDG). LC-PUFA biosynthesis is considered to start from oleic acid (18:1n9). EPA can be synthesized via a series of desaturation and elongation steps occurring at the endoplasmic reticulum and newly synthesized EPA is then imported into the plastids for incorporation into galactolipids via an unknown route. The basis for the flux of EPA is fundamental to understanding LC-PUFA biosynthesis in diatoms. We used P. tricornutum to study acyl modifying activities, upstream of 18:1n9, on subsequent LC-PUFA biosynthesis. We identified the gene coding for the plastidial acyl carrier protein Δ9-desaturase, a key enzyme in fatty acid modification and analysed the impact of overexpression and knock out of this gene on glycerolipid metabolism. This revealed a previously unknown role of this soluble desaturase in EPA synthesis and production of TAG. This study provides further insight into the distinctive nature of lipid metabolism in the marine diatom P. tricornutum and suggests additional approaches for tailoring oil composition in microalgae.