Algal lipids are the focus of intensive research because they are potential sources of biodiesel. However, most algae produce neutral lipids only under stress conditions. Here, we report that treatment with Brefeldin A (BFA), a chemical inducer of ER stress, rapidly triggers lipid droplet (LD) formation in two different microalgal species, Chlamydomonas reinhardtii and Chlorella vulgaris. LD staining using Nile red revealed that BFA-treated algal cells exhibited many more fluorescent bodies than control cells. Lipid analyses based on thin layer chromatography and gas chromatography revealed that the additional lipids formed upon BFA treatment were mainly triacylglycerols (TAGs). The increase in TAG accumulation was accompanied by a decrease in the betaine lipid diacylglyceryl N,N,N-trimethylhomoserine (DGTS), a major component of the extraplastidic membrane lipids in Chlamydomonas, suggesting that at least some of the TAGs were assembled from the degradation products of membrane lipids. Interestingly, BFA induced TAG accumulation in the Chlamydomonas cells regardless of the presence or absence of an acetate or nitrogen source in the medium. This effect of BFA in Chlamydomonas cells seems to be due to BFA-induced ER stress, as supported by the induction of three homologs of ER stress marker genes by the drug. Together, these results suggest that ER stress rapidly triggers TAG accumulation in two green microalgae, C. reinhardtii and C. vulgaris. A further investigation of the link between ER stress and TAG synthesis may yield an efficient means of producing biofuel from algae.