The study investigated the effects of replacing soybean meal (SBM) with slow-release urea (SRU) on milk production, milk composition, and rumen fermentation of Holstein dairy cows. Sixteen Holstein cows weighing between 550 and 680 kg in mid lactation were randomly assigned to four dietary treatments in a 12-week study. The treatments consisted of T1: a diet containing 16.7% crude protein (CP), T2: T1 with 0.5% SRU replacing plant protein, T3: T1 with 0.75% SRU replacing plant protein, and T4: T1 with 1.00% SRU replacing plant protein. Animals were fed three times a day with feed being offered ad libitum. Dry matter intake (DMI) and average daily gain (ADG) were not affected by the level of SRU. Feeding SRU did not affect milk production and milk composition significantly, but milk fat and milk urea nitrogen (MUN) levels were increased. Significant differences were observed in ruminal volatile fatty acid (VFA) concentration. Feeding SRU increased butyrate concentration with no significant effects on concentrations of acetate or propionate. Significant differences were observed in cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and nonesterified fatty acid (NEFA) concentrations, but glucose, very-low-density lipoprotein (VLDL) and β-hydroxybutyrate (BHB) levels were not affected significantly by the treatments. Thus, feeding SRU altered the release rate of ammonia and provided more ammonia nitrogen (NH3-N) for microbial protein synthesis in the rumen.