PF‐05221304 is a liver‐targeted inhibitor of acetyl‐CoA carboxylase, an enzyme that catalyzes the first committed step in de novo lipogenesis (DNL). This first‐in‐human study investigated safety/tolerability and pharmacokinetics of single and multiple ascending oral PF‐05221304 doses, and fructose‐stimulated DNL inhibition with repeated oral doses. Healthy subjects (n = 96) received single (1‐240 mg) or repeated (2‐200 mg daily) doses for 14 days or single 100‐mg doses with and without food. PF‐05221304 was well tolerated at all doses. Repeated PF‐05221304 doses inhibited hepatic DNL in a dose‐dependent manner, with near‐complete inhibition seen at higher doses. With doses yielding ≥90% DNL inhibition, asymptomatic increases in fasting/postprandial serum triglyceride levels (≥40 mg/day) and declines in platelet count (≥60 mg/day) occurred; these were not observed at ≤80% DNL inhibition. Steady‐state pharmacokinetics generally increased dose‐proportionally, with a half‐life of 14‐18 hours and a minimal food effect on plasma exposure. The observed safety and tolerability, pharmacokinetics, and pharmacodynamics support the continued evaluation of PF‐05221304 for the treatment of nonalcoholic steatohepatitis.