Comparative gene identification-58 (CGI-58) is an activating protein of triacylglycerol (TAG) lipase. It has a variety of catalytic activities whereby it may play different roles in diverse organisms. In this study, a homolog of CGI-58 in Phaeodactylum tricornutum (PtCGI-58) was identified. PtCGI-58 was localized in mitochondria by GFP fusion protein analysis, which is different from the reported subcellular localization of CGI-58 in animals and plants. Respectively, PtCGI-58 overexpression resulted in increased neutral lipid content and TAG accumulation by 42-46% and 21-32%. Likewise, it also increased the relative content of eicosapentaenoic acid (EPA), and in particular, the EPA content in TAGs almost doubled. Transcript levels of genes involved in de novo fatty acid synthesis and mitochondrial β-oxidation were significantly upregulated in PtCGI-58 overexpression strains compared with wild-type cells. Our findings suggest that PtCGI-58 may mediate the breakdown of lipids in mitochondria and the recycling of acyl chains derived from mitochondrial β-oxidation into TAG biosynthesis. Moreover, this study potentially illuminates new functions for CGI-58 in lipid homeostasis and provides a strategy to enrich EPA in algal TAGs.