Phenylalanine fermentation byproduct (PFB) is generated by purifying phenylalanine products from microbial fermentation in the food industry. We examined the function of PFB as an ensiling agent to improve the silage fermentation and nutritive value of forage crops. We investigated the fermentation quality and rumen degradability of whole crop barley (Hordeum vulgare L.) silage in situ. We ensiled this type of silage in a laboratory scale using five compositional ratios (v/v) of PFB (20%, 40%, 50%, 60% and 80%) and a mixture of sugarcane molasses (SCM). Controls included the same volumes of water as the mixture of sugarcane molasses. The following strains Lactococcus lactis ssp. lactis (IFO12007), Leuconostoc mesenteroides ssp. mesenteroides (IFO12060), Lactobaccillus plantarum (IF014713), Pedioccocus acidilactici (IFO3858), Clostridium butyricum (IFO3858) and silage yeast (strain MY; separated from whole crop rice silage in this laboratory) were cultivated at 30°C in the recommended media containing a substituted carbon source depending on the compositional ratios of PFB and SCM mixtures. The growth of these strains was determined by measuring the optical density at 600 nm (OD600). The growth of the lactic acid bacteria significantly (P < 0.001) increased in the presence of all PFB additives as compared with the control. The growth of C. butyricum was significantly increased, whereas that of silage yeast was decreased under all conditions. The concentrations of lactate and total organic acids were significantly higher (P < 0.001) whereas those of acetate were lower in all PFB silages. These results indicated that PFB improved the silage fermentation of forage crops. With leaf and stem fractions, the neutral detergent fiber (NDF) content was significantly (P < 0.001) decreased by the PFB additive and the rumen degradability of DM and NDF in situ was also significantly increased (P < 0.01 and P < 0.001, respectively).