Cell spheroids are applied in various fields of research, such as the fabrication of three-dimensional artificial tissues in vitro, disease modeling, stem cell research, regenerative therapy, and biotechnology. A preclinical 3D culture model of primary human gingival fibroblasts free of external factors and/or chemical inducers is presented herein. The ultrastructure of the spheroids was characterized to establish a cellular model for the study of periodontal tissue regeneration. The liquid overlay technique was used with agarose to generate spheroids. Fibroblasts in 2D culture and cell spheroids were characterized by immunofluorescence, and cell spheroids were characterized by optical and scanning electron microscopy, energy-dispersive X-ray spectroscopy, backscattered electrons, and Fourier transform infrared spectroscopy. Ostegenic related genes were analyzed by RT-qPCR. Gingival fibroblasts formed spheroids spontaneously and showed amorphous calcium phosphate nanoparticle deposits on their surface. The results suggest that human gingival fibroblasts have an intrinsic potential to generate a mineralized niche in 3D culture.