Carbon isotopic fractionation was studied during the development of the diatom Chaetoceros muelleri grown in batch culture with 13C-depleted CO2 addition. Cellular and growth parameters and isotopic composition of dissolved inorganic carbon and particulate organic carbon were monitored every two days, while the content and isotopic composition of individual fatty acid in polar lipid and neutral lipid were measured on the 5th day (end of exponential phase), 10th and 14th days (stationary phase). Continuous addition of petrochemical CO2 to the algae led to a rapid and strong modification of dissolved inorganic carbon isotopic composition with cascading effects on particulate organic carbon and fatty acid isotopic compositions. Carbon isotope fractionation in Chaetoceros muelleri ranged from 17‰ to 25‰ and changed according to culture ages. Isotopic fractionation into fatty acids, overall, was similar between polar and neutral lipids, and was systematically higher than in particulate organic carbon. At the end of the exponential growth phase, the isotopic composition of individual fatty acids varied from −51.3‰ to −58.4‰. At this culture age, large differences in the isotopic compositions between fatty acids were observed. Polyunsaturated fatty acids such as 16:3n-4, 18:4n-3, and 20:5n-3 were more strongly 13C-depleted than other fatty acids such as 14:0, 16:0, 16:1n-7 or 18:1n-9. These results showed how isotopic effects occur during the desaturation and elongation phases. Such isotopic effects were also supported by the lower δ13C of averaged δ13C of saturated fatty acids and monounsaturated fatty acids as compared to those of polyunsaturated fatty acids. However, during the stationary phase, fatty acid isotopic compositions were less variable and closer to particulate organic carbon, while saturated and monounsaturated fatty acids were more depleted than polyunsaturated fatty acids. Our study underlined the importance of consideration of phytoplankton physiological status when conducting ecological and biogeochemical studies as they appeared to strongly control phytoplankton carbon isotopic composition.