Phaeodactylum tricornutum is a well-studied oleaginous model diatom with high triacylglycerol (TAG) and omega-3 eicosapentaenoic acid (EPA) contents, however, only a small portion of EPA is transferred from the polar lipids to TAG through phospholipid:diacylglycerol acyltransferase (PDAT). In this study, a Nannochloropsis oceanica PDAT (NoPDAT) was heterologously expressed in P. tricornutum to increase the EPA content in TAG. NoPDAT heterologous expression slightly disturbed the growth of the transgenic lines, but enhanced the TAG content by 16–18 %. Considering the localization of NoPDAT in P. tricornutum after heterologous expression together with our lipidomic results, NoPDAT is more likely to utilize phosphatidylcholine and phosphatidylethanolamine on the chloroplast endoplasmic reticulum membrane as the acyl donors. Especially, NoPDAT transferred the EPA acyl moiety from the sn-2 position of the two above-mentioned phospholipids to the sn-3 position of TAG, resulting in the increase of two major EPA-containing TAG molecules (16:1/16:0/20:5 and 16:1/16:1/20:5). Correspondingly, two major 16:0-containing TAG molecules (16,0/16:1/16:1 and 16:0/16:0/16:1) decreased in the transgenic strains. In conclusion, our attempt to express NoPDAT in P. tricornutum increases the EPA content in TAG by approximately one-fold, and demonstrates the versatility of diatom lipid metabolism and acyl editing.