Mitragynine (MTR), the main indole alkaloid of the well-known plant kratom (Mitragyna speciosa), is one of the most studied natural products nowadays, due to its remarkable biological effects. It is a partial agonist on the opioid receptors, and as such relieves pain without the well-known side-effects of the opioids applied in the clinical practice. MTR and its derivatives therefore became novel candidates for drug development. The poor aqueous solubility and low bioavailability of drugs are often improved by cyclodextrins (CyDs) as excipients through host-guest type complex formation. Among the wide variety of CyDs, sulfobutylether-beta-cyclodextrin (SBEbetaCyD) is frequently used and official in the European and U.S. Pharmacopoeia. Herein, the host-guest complexation of MTR with betaCyD and SBEbetaCyD was studied using chiroptical and NMR spectroscopy. It was found by NMR measurements that MTR forms a rather weak (logbeta(11) = 0.8) 1:1 host-guest complex with betaCyD, while the co-existence of the 2MTR鈭橲BEbetaCyD and MTR鈭橲BEbetaCyD species was deducted from (1)H NMR titrations in the millimolar MTR concentration range. Sulfobutylation of betaCyD significantly enhanced the affinity towards MTR. The structure of the formed inclusion complex was extensively studied by circular dichroism spectroscopy and 2D ROESY NMR. The insertion of the indole moiety was confirmed by both techniques.