Nicotinoids are agonists of the acetylcholine receptor (nAChR) and play important biochemical and pharmacological roles. Here we report on the structural and conformational landscape of cotinine, and compare its molecular properties with the nicotine prototype, from which it only differs in the addition of a carbonyl group. The investigation of cotinine included a theoretical survey of the effects of rotamerization of the pyridine moiety, the puckering of the pyrrolidinone ring and the internal rotation of the methyl group. The experimental work examined the rotational spectrum of the molecule in a supersonic expansion, using both broadband chirped-pulse excitation techniques and cavity microwave spectrometers. Two conformers were observed for cotinine, and the fine and hyperfine structures arising from the two quadrupolar 14N nuclei and the methyl internal rotor were fully analyzed. The two observed conformers share the same twisted conformation of the five-membered ring, but differ in a ~180o rotamerization around the C-C bond connecting the two cycles. The barrier heights for the internal rotation of the methyl top in cotinine (4.55(4) and 4.64(3) kJ/mol, respectively) are much lower than in nicotine (estimated in 16.5 kJ/mol). The combination of different intramolecular electronic effects, hydrogen bonding and possible binding differences to receptor molecules arising from the carbonyl group could explain the lower affinity of cotinine for nAChRs.