Clostridium autoethanogenumprotein (CAP) is an economical and alternative protein source. Here, three experimental diets were formulated with CAP replacing 0% (CAP-0), 30% (CAP-30), and 60% (CAP-60) of fishmeal to investigate the alterations of structure integrity, fatty acids profiles, and lipid metabolism in the muscle of pearl gentian grouper. With increasing levels of CAP substitution, the percentages of 16 : 0 or 18 : 0 were decreased in triglycerides (TG) and diacylglycerols (DG); 18 : 1 or 18 : 2 was increased at thesn−1andsn−2positions in phosphatidylethanolamines; 20 : 5n−3 was increased in TG and DG. The phosphatidylcholines (PC) (18 : 3/20 : 5), PC(22 : 6/17 : 1), and sphingomyelins (d19 : 0/24 : 4) were identified as potential lipid biomarkers between CAP treatments. The CAP-30 treatment enhanced lipolysis and lipogenesis, while the CAP-60 treatment inhibited lipogenesis. In conclusion, fishmeal replacement with CAP affected the lipid characteristics and lipid metabolism, whereas it did not affect the structural integrity and fatty acids profiles in the muscle of pearl gentian grouper.