Aim: To assess the effect of protein-containing toothpastes on the progression of dental erosion in situ (with pellicle) and in vitro (without pellicle). Methods: A combined split-mouth (extraoral water or toothpaste brushing) and crossover (type of toothpaste) setup was used. Two protein-containing (high/low concentrations of colostrum) and one nonprotein (placebo) toothpaste were investigated. Sixteen volunteers wore intraoral appliances containing 2 human enamel samples on 3 afternoons for pellicle growth during 90 min. One enamel sample was brushed for 5 s with one of the three toothpastes and subsequently exposed to a slurry of the corresponding toothpaste for 2 min. The other sample was exposed to water. Both samples were subsequently exposed to citric acid (extraorally). Loss of calcium and inorganic phosphate were determined. The same sequence of exposures was applied to 16 enamel samples in an in vitro setup without pellicle. Results: With the in situ-formed pellicle, all toothpastes significantly reduced calcium loss compared to water brushing, although no significant differences were found among toothpastes (p = 0.073). For the loss of phosphate, a significant reduction could be found with the use of the high-protein toothpaste compared to the nonprotein toothpaste. Overall there were only slight differences between the toothpastes. Toothpaste effects were less clear in the in vitro experiment. Conclusion: The addition of proteins to toothpaste shows some promise for the prevention of erosion. Further research is needed to investigate the performance of the protein-containing toothpastes in longer in situ studies with regard to erosive wear.