Open AccessThe objective of this study was to evaluate intake, microbial protein synthesis, and nitrogen balance in lambs fed diets containing 0.00, 12.50, and 25.00% mulberry hay substituting the concentrate. Twentyfour Ile de France lambs at approximately 60 days of age, with a body weight of 15.48 ± 0.07 kg, were confined in individual stalls and slaughtered upon reaching 32 kg body weight. Only the intakes of ether extract and metabolizable energy decreased linearly (P<0.05) as the participation of mulberry hay in the concentrate was increased. Nitrogen in the feces, expressed in g day–1 and g kg0.75 day–1, increased linearly (P<0.05), whereas excretions (mmol day–1) of allantoin, total purines, and absorbed microbial purines, microbial production (g day–1) expressed in microbial nitrogen and microbial crude protein, and microbial efficiency expressed in g MN kg–1 RDOM–1 and g MP kg–1 of TDN decreased linearly as the level of hay in the diet was increased. Mulberry hay substituting the concentrate in diets for feedlot lambs reduces the microbial protein synthesis.