Three rumen-cannulated Holstein steers were fed three diets, each with a different synchrony index (SI) (LS: 0.77, MS: 0.81, and HS: 0.83), in order to examine the effect of diet on rumen fermentation, nitrogen balance, and microbial protein synthesis. Synchrony index was calculated based on the carbohydrate and crude protein fractions of each ingredient and their degradation rates. Feeding the steers diets with different SIs did not influence dry matter, crude protein, NDF, or ADF digestibility. The concentrations of total and individual VFA in the rumens of steers that were fed the two higher-SI diets were higher than in those fed the low-SI diet (p<0.05), but there was no significant difference between the two higher-SI diets. One hour after feeding, steers on the LS diet had lower ruminal pHs than did those fed the MS or HS diets (p<0.05), and animals on the LS diet generally showed higher ruminal $NH_3$-N levels than did animals on the other diets, with the 4-h post-feeding difference being significant (p<0.05). Steers receiving the LS diet excreted more nitrogen (N) in their urine than did those on the two higher-SI diets (p<0.05), and the total N excretion of those on the LS diet was also higher (p<0.05). Microbial N levels calculated from the concentration of urinary purine derivatives were generally higher when the SI was higher, with the highest microbial protein synthesis being produced by steers on the HS diet (p<0.05). In conclusion, in the current study, ingestion of a synchronous diet by Holstein steers improved microbial protein synthesis and VFA production and decreased total N output.