To evaluate the protective effect of phytosphingosine (PHS) against staining on dental enamel. Ninety-six specimens of bovine teeth (6 mm × 6 mm × 2 mm) were cut, and initial color (Easyshade, VITA), microhardness (HMV-2, Shimadzu) and fluorescence (Matlabs software, Matworks) measurements were performed. Specimens were separated into four groups according to the treatments: Distilled water (control); Human saliva (HS); PHS; PHS + HS. Specimens (n = 6) were submitted to staining procedures: Distilled water (immersion for 30 days-control); Coffee (15 minutes, twice a day/for 15 days); Black tea (as described for Coffee) and cigarette smoke (20 cigarettes/sample). Final measurements were performed, and data were analyzed (Color-CIEDE2000, fluorescence-2-way ANOVA, Tukey, and microhardness-Kruskal-Wallis Dunn, P < .05). Coffee caused the highest color change (ΔE00 ), followed by black tea, regardless of the treatment employed. Distilled water and cigarette smoke produced similar color changes (P > .05) for the groups control (water = 1.0 + - 0.5/ cigarette = 2.3 0.3) and PHS (water = 0.8 0.4/ cigarette = 2.3 0.4). PHS + HS demonstrated intermediate means than PHS and HS when stained with coffee and tea. After treatments, the least fluorescence alterations occurred for the groups treated with distilled water and cigarette, regardless of the treatment, with no difference (P > .05) between them. There was a significant difference (P < .05) on microhardness between all the groups, as PHS + HS > PHS > HS > Distilled water. It was concluded that PHS treatment did not protect the staining of the enamel by coffee and tea, but increased the microhardness, both in the presence and absence of a salivary pellicle. Phytoshingosine is a novel agent and considered a promising component for anti-biofilm and anti-erosion properties by the formation of a diffusion barrier on the dental enamel. In line, PHS might be considered for anti-staining purposes.