Acetyl-coenzyme A carboxylases (ACCs) catalyze the initial reaction of fatty acid (FA) biosynthesis. The marine diatom Phaeodactylum tricornutum has two nuclear-encoded ACCs (PtACC1 (Phatr3_EG01955) and PtACC2 (Phatr3_J55209)), both which are homomeric and predicted to be localized in the plastids and the cytosol, respectively. In this study, we focused on stromal ACC1 by constructing P. tricornutum strains expressing GFP-tagged PtACC1 (ACCG strains) and confirmed that PtACC1 was localized in or around the pyrenoid. Here, we showed that unsaturated FAs (UFAs) composing the thylakoid membrane lipids increased in PtACC1 strains grown under high light conditions (190 µmol photons m−2 s−1), and that the content of triacylglycerol (TAG) and unsaturation ratios in TAG increased under oxidative stresses (with added 50 µM H2O2). ACCG strains showed faster growth rates than wild type under high light and/or oxidative stress conditions. These results suggest that cell proliferation is maintained by an accelerated recovery of PSII due to the increased UFAs in the thylakoid membrane in ACCG strains grown in high light, and that increased UFAs in ACCG cells enhanced the tolerance to oxidative stresses presumably due to the increased scavenging capacity of UFAs against reactive oxygen species. The introduction of plastidic ACC resulted in stimulating supply of UFAs to specific lipids that in turn enhanced tolerance to various stresses.