A growing body of evidence has suggested that 3D cell culture systems, in contrast to the 2D systems, represent more accurately the actual microenvironment where cells reside in tissues. Therefore, researchers have concentrated on developing realistic and sophisticated in-vitro 3D cell culture models that actually resemble the complex environment of native tissues. Calixarenes, similar to a ring basket, represent a third generation of supramolecular hosts could be promising candidates in this regard. Due to having a cyclic structure and large surface area, calixarenes, can be functionalized easily with polar and apolar groups, be a good carrier for cations, anions and neutral molecules. Electrospinning of p-tert-butylcalix[4]arene nanofibers with different functional groups were performed and their endothelial (Caco-2) and fibroblastic (L-929) cytocompatibility behaviour on cell adhesion function were examined. Cell attachment kinetics revealed that the Caco-2 and L-929 attached to the nanofibers functionalized with pyridinum at position 3 at the same rate as to tissue culture plates. In this report, for the first time in literature, it was shown that 3D cultured systems designed with calixarene nanofibers provide excellent in vitro models, allowing the study of cellular responses in a setting that resembles in vivo environments.