Indolizidine (−)-235B′ is a particularly interesting natural product, as it is the currently known, most potent and subtype-selective open-channel blocker of the α4β2 nicotinic acetylcholine receptor (nAChR). In the current study, extensive first-principles electronic structure calculations have been carried out in order to determine the stable molecular conformations and their relative free energies of the protonated and deprotonated states of (−)-235B′ in the gas phase, in chloroform, and in aqueous solution. The ¹H and ¹³C NMR chemical shifts calculated using the computationally determined dominant molecular conformation of the deprotonated state are all consistent with available experimental NMR spectra of (−)-235B′ in chloroform, which suggests that the computationally determined molecular conformations are reasonable. Our computational results reveal for the first time that two geminal H atoms on carbon-3 (C3) of (−)-235B′ have remarkably different chemical shifts (i.e., 3.24 and 2.03 ppm). The computational results help one to better understand and analyze the experimental ¹H NMR spectra of (−)-235B′. The finding of remarkably different chemical shifts of two C3 geminal H atoms in a certain molecular conformation of (−)-235B′ may also be valuable in analysis of NMR spectra of other related ring-containing compounds. In addition, the pK _a of (−)-235B′ in aqueous solution is predicted to be ~9.7. All of the computational results provide a solid basis for future studies of the microscopic and phenomenological binding of various receptor proteins with the protonated and deprotonated structures of this unique open-channel blocker of α4β2 nAChRs. This computational study also demonstrates how one can appropriately use computational modeling and spectroscopic analysis to address the structural and spectroscopic problems that cannot be addressed by experiments alone.