Abstract: Currently, production of biodiesel by microalgae has been regarded as a promising source of renewable energy. However, the understanding of oil biosynthesis mechanisms in micro-algae is limited. Phospholipid: diacylglycerol acyltransferase catalyzes phospholipid and diacylglycerol to produce triglyceride, a key reaction in triglyceride synthesis. In this study, we cloned a fragment of Phospholipid: diacylglycerol acyltransferase homologous gene 3 CrPDAT3 in Chlamydomonas, which was then used to construct a CrPDAT3 RNAi interference vector and transferred into Chlamydomonas. The results showed that the growth rate of transgenic algae strain was declined. At the same time, the oil content was decreased by 14.65%-45.15%, showing that the CrPDAT3 playing an important role in oil biosynthesis.