Sulphur dioxide (SO) and fluoride are among the most common environmental pollutants affecting human health, and both co-exist in areas predominantly consuming coal. It is vital to analyse the combined toxicity of SO and fluoride, and their effects on health and the underlying mechanisms of their co-exposure have not yet been adequately assessed. In the present study, we used ICR mice and LS8 cells to investigate the toxicity of SO and fluoride exposure to the enamel, alone or in combination. Factorial design analysis was used to reveal the combined toxicity in vitro and in vivo. Co-exposure to SO and fluoride exacerbated enamel injury, resulting in more severe hypomineralization of incisor, and enamel structure disorders in mice, and could induce the accumulation of protein residue in the matrix of the enamel. Amelogenin expression was increased upon exposure to SO and fluoride, but enamel matrix proteases were not affected. Consistent with our in vivo results, co-exposure of SO and fluoride aggravated amelogenin expression in LS8 cells, and increased the YAP and RUNX2 levels. Co-exposure to SO and fluoride resulted in greater toxicity than individual exposure, both in vitro and in vivo, indicating that residents of areas exposed to SO and fluoride may have an increased risk of developing enamel damage.