The aim of this study was to determine the effect of additional nucleus insertion and its corresponding position on the shell growth of Pteria penguin as well as the thickness of the resulting mabé pearls. The treatment applied was the insertions of nucleus: A (one into the left shell), B (two into the left shell), and C (one into each sides of the shell). After 4 mo of culture, the growth and structure of the layers in the pearl were observed. The development of pearl layers was observed using scanning electron microscope. Results show that the number of nuclei implanted in the shell has no significant impact on shell growth or thickness as well as the number of aragonite layers of mabé pearls. Growth data show changes in oyster shell height, length, and thickness of 11.55–14.78, 18.48–22.05, and 2.59–6.14 mm, respectively, for all treatments. The average thickness of nacre was 0.29–0.33 and 0.19–0.22 µm for the base and top of resulting mabé pearls, respectively. In general, the addition of two nuclei in a single pearl oyster can be used to increase mabé pearl production in the coastal areas of Palabusa Bay, Southeast Sulawesi, Indonesia, which is suitable for the growth of pearl oysters.