Acetyl-CoA carboxylase (ACCase) catalyzes the first and rate-limiting step in de novo fatty acid synthesis in the chloroplast. ACCase activity has thus been subjected to positive and negative regulations. Among negative regulators, one finds the pII, BADC and the carboxyltransferase interactor (CTI) in Arabidopsis thaliana. However, we know little about the regulation of fatty acid synthesis in algae. In this study, we identified only one homolog of the plant CTI in Chlamydomonas and show that the Chlamydomonas CrCTI1 indeed interacts with the Chlamydomonas alhpa-carboxyltransferase (CT) ACCase subunit by yeast two-hybrid protein-protein interaction assay. The Chlamydomonas CrCTI1 knocked-out mutants, obtained from the CRISPR-Cas9 mutagenesis, accumulated higher amount of total fatty acids and stored more triacylglycerols (TAGs) in lipid droplets, without affecting membrane lipids. The TAG phenotype of the crcti1 mutants was not influenced by light, but rather is affected by trophic styles. Moreover, cell cultivation under heterotrophic conditions in darkness revealed the crucial function of CrCTI1 in homeostasis between lipid accumulation and cell growth. Comparative proteomics analysis of the crcti1 mutants and WT suggested a role for CrCTI1 in maintaining carbon flux and redox balance under different carbon availability. Taken together, this study reveals the occurrence and function of a negative regulator of fatty acid synthesis in algae and provides a molecular brick that can be used for genetic engineering of microalgae for biotechnology purposes.