Rifampicin (Rif) is a broad spectrum antibiotic used as a first line agent in the treatment of mycobacterial infections. However, its low solubility and reduced stability in water limit its bioavailability, thus requiring the use of complex formulations. Here, we present a systematic study of Rif in complex with a methylated cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin (DIMEB), in phosphate buffer using a combination of nuclear magnetic resonance (NMR) and steady-state UV–vis spectroscopic methods. An increase in the stability and solubility of Rif in complex with DIMEB was observed in buffered solutions (phosphate, PBS). At neutral pH the presence of three distinguishable binding sites was revealed, demonstrating that DIMEB forms predominantly a stable 1:1 (K ˜ 3000 M−1) complex at the piperazine site of Rif, while at acidic pH the binding constant decreases significantly (K ˜ 400 M−1) due to protonation of the piperazine, thus inducing a release of Rif. The reported results provide new and relevant information for the stability and solubility of Rif in aqueous solution when forming a complex with DIMEB. Furthermore they contribute to clarify Rif interactions with cyclodextrin carriers, thus providing the basis for the development of new methylated cyclodextrin that can efficiently encapsulate and deliver Rif and derivatives of its family.