Cyclosporine (CsA) has a selective property of suppressing various T-lymphocyte functions. This is of utmost importance in preventing allograft rejection by several organ transplantations, as well as in the treatment of systemic and local autoimmune disorders. However, the poor water solubility of CsA can be a major hurdle for its absorption into the blood stream, which leads to low bioavailability and thus less efficacy. The aim of this study was to prepare, characterize, and evaluate in vitro as well as in vivo, the potential of the innovative CsA drug delivery system. The latter contains CsA in spherical amorphous solid dispersion (SASD) which is embedded in an original α-cyclodextrin and β-cyclodextrin polymer mixture (Poly-αβ-CD) as a multifunctional amorphous carrier. The new developed SASD formulation showed that CsA was molecularly dispersed in αβ-cyclodextrins in an amorphous form, as was confirmed by physicochemical characterization studies. Interestingly, the peptide secondary structure, and thus, the drug activity was not impacted by the preparation of SASD as was shown by circular dichroism. Furthermore, the in vitro CsA release profile kinetics was almost identical to the commercially available product Neoral^®. This study presents the first in vivo proof-of-concept for a novel drug delivery system based on Poly-αβ-CD containing CsA, with SASD allowing for increased bioavailibility. The pharmacokinetic parameters of cyclosporine A from the spherical spray-dried dispersion formulation was demonstrated in a “rat” animal model. For comparison, the commercially available Neoral® was studied. Importantly, the pharmacokinetic parameters were improved by extending T_max from 2 to 3 h after the oral administration in rats, and eventually preventing the enterohepatic circulation. All these results clearly demonstrate the improved pharmacokinetic parameters and enhanced bioavailability of CsA in the new developed drug delivery system. These data demonstrated the superiority of the newly developed Poly-αβ-CD formulation for oral administration of the poorly soluble CsA in vivo without altering its secondary structure. Poly-αβ-CD can be a very useful tool for the oral administration of poorly water-soluble drugs.