In the prior study, the dyeing behaviors of Milling Cyanine 5R on silk fiber in aqueous dyeing liquor including small amount of organic compounds were investigated. The most effective compound was acetophenone which increased dye uptake as well as dyeing rate. In this study, the role of the acetophenone in dyeing of silk with Milling Cyanine 5R was studied. By addition of acetophenone into the dye solution, the molar absorptivity of Milling Cyanine 5R increased, and the wavelength of maximum absorption was shifted to longer wavelength, namely bathochromic shifted. This shows that the acetophenone prevents the dye to aggregate, and increases the number of monomeric dye molecule relative to the dye solution without acetophenone. This fact was also confirmed by the increase of the permeation rate of the dye through cellulose semipermeable membrane from the dye solution including the acetophenone. From these results, the acetophenone acts as a prohibitor of dye aggregation, increases the number of monomeric dye molecules even at relatively low temperature, and makes the dyes penetrate into the fiber easier.