The conformational landscape of the alkaloid anabasine (neonicotine) has been investigated by using rotational spectroscopy and ab initio calculations. The results allow a detailed comparison of the structural properties of the prototype piperidinic and pyrrolidinic nicotinoids (anabasine vs. nicotine). Anabasine adopts two most stable conformations in isolation conditions, for which we determined accurate rotational and nuclear quadrupole coupling parameters. The preferred conformations are characterized by an equatorial pyridine moiety and additional N-H equatorial stereochemistry at the piperidine ring (eq-eq; eq=equatorial). The two rings of anabasine are close to a bisecting arrangement, with the observed conformations differing by an approximately 180 degrees rotation of the pyridine subunit, denoted either syn or anti. The preference of anabasine for the eq-eq-syn conformation has been established by relative intensity measurements (syn/anti approximately 5(2)). The conformational preferences of free anabasine are directed by a weak N...H-C hydrogen bond interaction between the nitrogen lone pair at piperidine and the closest C-H bond in pyridine, with N...H distances ranging from 2.686 (syn) to 2.667 A (anti). Supporting ab initio calculations by using MP2 and the recent M05-2X density functional are provided, evaluating the predictive performance of both methods.