It has been theoretically predicted that spherical particles added to the smectic phase formed by rod-like particles segregate between the smetic layers and significantly enhance the stability of the smectic phase due to an entropic effect based on the steric repulsion between the particles, which is referred to as the depletion effect. In this study, we describe the first experimental verification of the theoretical prediction, in which the binary mixtures of the rod-like polymer, poly[n-decyl-(S)-2-methylbutylsilane] (PDMS), with narrow molecular weight distributions, and a spherical branched alkane, squalane, were investigated by synchrotron radiation small- and wide-angle X-ray scattering (SR-SAXS and SR-WAXS) and atomic force microscopy (AFM) observations and found to reproduce the predicted segregation between the smectic layers. [DOI: 10.1380/ejssnt.2015.121]