Dopant segregation at grain boundaries (GBs) in ceramics has been widely reported, while whether similar segregation behavior occurs in glass-ceramics remains unknown. The distribution of dopant in glass-ceramics may be totally different due to the existence of glass phase. This study examines the distribution of Y3+ ions in a ZrO2-SiO2 glass-ceramic. Two samples were prepared by hot pressing, yttrium oxide-doped, and undoped 65 mol% ZrO2-35 mol% SiO2 nanocrystalline glass-ceramics (NCGCs). The NCGCs had the same microstructure, that is, ZrO2 nanoparticles (NPs) embedded in an amorphous SiO2 matrix. XRD results showed that the undoped NCGC was composed of 20.9 wt% (weight percentage) monoclinic ZrO2 (m-ZrO2) and 79.1 wt% tetragonal ZrO2 (t-ZrO2), while the yttrium oxide-doped NCGC was composed of 9.6 wt% m-ZrO2 and 90.4 wt% t-ZrO2. X-ray energy-dispersive spectrometry (EDS) results in scanning electron transmission microscopy (STEM) mode demonstrated that Y3+ ions segregated both on the surface of ZrO2 NPs and within the thin intergranular glass film (with a thickness of approximately 7 angstrom) between ZrO2 NPs in the yttrium oxide-doped NCGC. Interestingly, no obvious Y signals were detected in the amorphous SiO2 matrix. Density functional theory calculation results showed that Y3+ ions had a strong segregation tendency in the GB area and the segregation of Y3+ ions increased the work of separation of GB layer. These findings provide new understanding of the segregation behavior of dopant in glass-ceramics, which may offer useful guidance for other researchers to tailor the properties of glass-ceramics through GB engineering.