Membrane lipids play essential roles in regulating physiological properties in higher plants and algae. Monogalactosyldiacylglycerol (MGDG) is a major thylakoid membrane lipid, and it is an important source of polyunsaturated fatty acids for cells, plays a key role in the biogenesis of plastids, and maintains the function of the photosynthetic machinery. Several studies have indicated that the knockdown of MGDG synthase results in membrane lipid remodeling, albino seedlings, and changes in photosynthetic performance. However, the effects of MGDG synthase (MGD) inhibitors on lipids in macroalgae have not yet been clarified. Here, we characterized the effects of MGD inhibitors (ortho‐phenanthroline and N‐ethylmaleimide) on the composition of the fatty acids observed in MGDG and digalactosyldiacylglycerol (DGDG) in Gracilariopsis lemaneiformis using electrospray ionization–mass spectrometry. The most abundant MGDG species contained 16:0/18:1 (sn‐1/sn‐2) fatty acids, and the most dominant DGDG species contained 20:5/16:0 (sn‐1/sn‐2) fatty acids. Measurements of photosynthetic pigments and photosynthetic parameters revealed that photosynthesis of G. lemaneiformis was impaired. Principal component analysis and Spearman's correlation analysis revealed interactions between specific MGDG structural composition patterns and key metabolites involved in photosynthesis, indicating that 20:4/16:0 (sn‐1/sn‐2) MGDG and 16:0/18:1 (sn‐1/sn‐2) MGDG affect the structure and function of phycobilisomes and thus the color of G. lemaneiformis. Three genes (GlMGD1, GlMGD2, and GlMGD3) were cloned and identified. The addition of N‐ethylmaleimide to G. lemaneiformis did not affect the abundance of GlMGD mRNA, and the abundance of transcripts was significantly decreased by ortho‐phenanthroline.