Carbon dioxide, the sole carbon source in phototrophic cultivation of microalgae, is the limiting factor for photosynthesis due to its low concentration in the atmosphere (0.04% v/v). We postulate that exposure to increased CO2 concentrations can increase the steady-state specific intracellular levels of carbon metabolic intermediates, including, the specific intracellular (si) levels of the precursor for fatty acid synthesis (acetyl CoA (AcCoA)), which in turn, appears to improve lipid accumulation. The effects of higher CO2 concentrations on Chlorella vulgaris were studied. At 2.6% v/v CO2, a 6-fold increase in volumetric lipid production was achieved. A 5.4-fold increase in the specific intracellular neutral lipid level (si-NL) from 9.6 to 52.3 mg triolein (TO)/g biomass was also obtained. Si-AcCoA was significantly elevated at 2.6% CO2, and showed a 41, 25 and 27% increase over the air-sparged controls in the lag, log and stationary phases, respectively. Further, we show a quantitative empirical relationship between si-AcCoA and si-NL, which provides a new outlook for the design of strategies to improve lipid accumulation. In addition, favorable biodiesel characteristics were also obtained at higher CO2. Increased si-AcCoA with increase in CO2 concentrations, a related increase in lipid levels, and a quantitative empirical relationship between them have not been reported in the literature thus far. © 2016 Elsevier B.V.