To evaluate whether protein changes in extracellular matrix of dental biofilm are a unique property of sucrose, this in situ study was conducted using as active control glucose and fructose, the sucrose monosaccharide constituents. Proteins were analyzed by two-dimensional electrophoresis followed by LC-MS/MS after trypsin digestion. Absence or lower abundance of calcium-binding proteins and higher abundance of prolactin-induced proteins were found in biofilm formed in the presence of sucrose or its monosaccharide constituents compared with water, the negative control group. The data suggest that besides sucrose, other dietary carbohydrates may also provoke a change in the protein profile of extracellular matrix of dental biofilm formed.