Background: This study focused on the interaction of arginine and fluoride on the remineralization of artificial enamel carious lesions in vitro. Methods: Human enamel blocks with artificial carious lesions were prepared and randomly divided into six treatment groups (n = 30 for each group): deionized water, arginine solution, NaF solution, arginine/NaF solution, arginine-free fluoride toothpaste slurry and arginine-containing fluoride toothpaste slurry. After a pH-cycling regimen for 10 days, subsets of specimens from each group were subjected to independent analysis, including surface microhardness (n = 8/30), cross-sectional microhardness (n = 8/30), enamel fluoride uptake (n = 8/30) and polarized light microscopy (n = 6/30). The samples tested for surface microhardness were further subjected to simulated plaque acid challenge before surface microhardness was remeasured. Results: Arginine solution promoted remineralization compared with deionized water control (p < 0.05). When used in combination with fluoride, arginine significantly increased fluoride uptake compared with fluoride alone (p < 0.05). In addition, lesions treated with arginine-containing toothpaste also showed superior fluoride uptake compared with those treated with conventional fluoride toothpaste (p < 0.05). Conclusions: Arginine promoted enamel fluoride uptake when used in combination with fluoride, thereby contributing to resistance of enamel to carious demineralization.