Background: Dental fluorosis is a condition resulting from excessive fluoride intake during tooth development, leading to abnormalities in enamel formation. This study aimed to investigate the impacts of transmembrane protein 16A (TMEM16A) on ameloblast proliferation, apoptosis, and the expression levels of associated genes in a dental fluorosis cell model under high-calcium conditions. Methods: Ameloblasts were isolated from two C57BL/6 male mice and treated with NaF (3.2 mmol/L) for 24 hours to establish the dental fluorosis cell model. Subsequently, ameloblasts were cultured in Dulbeccos modified eagle medium (DMEM) with varying calcium concentrations such as 2.0, 3.0, or 4.0 mmol/L, along with 10% fetal bovine serum, for 48 h, representing low, medium, and high calcium treatment groups, respectively. Control and NaF model groups were also included. Immunohistochemistry was used to identify the specific marker ameloblastin (AMBN) for ameloblasts. Cell proliferation was assessed using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, apoptosis was examined using TUNEL assay, and intracellular Ca²⁺ concentration was measured using fluorescent probes. Moreover, the impacts on the expression of apoptosis-related genes (Bcl2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), nuclear factor kappaB (NF-κB), nuclear factor kappa-Bp65 (NF-κBp65)), bone morphogenetic protein-2 (BMP-2), and TMEM16A were evaluated using quantitative real time polymerase chain reaction (qRT-PCR) and Western blot analysis. Additionally, the effects of the TMEM16A inhibitor T16Ainh-A01 on cell apoptosis and gene expression were investigated. Results: Compared to the NaF model group, calcium treatment significantly increased AMBN in ameloblasts, promoted cell proliferation (p < 0.01), reduced intracellular Ca²⁺ concentration (p < 0.01), and inhibited cell apoptosis. Moreover, calcium treatment substantially elevated the expression levels of B-cell lymphoma-2 (Bcl-2) and BMP-2 (p < 0.05), while suppressing the expression levels of Bcl2 associated X protein (Bax), nuclear factor kappa-Bp65 (NF-κBp65), and TMEM16A, with the highest efficacy observed in the high-calcium group (p < 0.05). Co-treatment with the TMEM16A inhibitor T16Ainh-A01 and high-dose calcium further enhanced cell proliferation (p < 0.01), reduced Ca²⁺ concentration (p < 0.05), and inhibited cell apoptosis. Additionally, T16Ainh-A01 significantly increased the expression levels of Bcl-2 and BMP-2, while suppressing Bax and NF-κBp65 expression (p < 0.05). Conclusion: Elevated levels of calcium and inhibition of TMEM16A expression can inhibit the apoptosis of ameloblasts caused by fluorosis by regulating the NF-κBp65 signaling pathway and apoptosis-related genes, potentially alleviating fluorosis-induced tooth damage. These findings offer a novel strategy for the prognosis and treatment of dental fluorosis.