Microalgae accumulate triacylglycerols (TAGs), a promising feedstock for biodiesel production, under unfavorable environmental or stress conditions for their growth. Our previous analyses revealed that only transcripts of CmGPAT1 and CmGPAT2, both encoding glycerol-3-phosphate acyltransferase, were increased among fatty acid and TAG synthesis genes under TAG accumulation conditions in the red alga Cyanidioschyzon merolae. In this study, to investigate the role of these proteins in TAG accumulation in C. merolae, we constructed FLAG-fused CmGPAT1 and CmGPAT2 overexpression strains. We found that CmGPAT1 overexpression resulted in marked accumulation of TAG even under normal growth conditions, with the maximum TAG productivity increased 56.1-fold compared with the control strain, without a negative impact on algal growth. The relative fatty acid composition of 18:2 in the TAGs and the sn-1/sn-3 positions were significantly increased compared with the control strain, suggesting that CmGPAT1 had a substrate preference for 18:2. Immunoblot analysis after cell fractionation and immunostaining analysis demonstrated that CmGPAT1 localizes in the endoplasmic reticulum (ER). These results indicate that the reaction catalyzed by the ER-localized CmGPAT1 is a rate-limiting step for TAG synthesis in C. merolae, and would be a potential target for improvement of TAG productivity in microalgae.