This experiment was designed to evaluate the effects of combining calcium salts of soybean oil fatty acids (CSFA) and two rumen degradable protein (RDP) levels on feed intake, ruminal fermentation, N metabolism, and performance of lactating cows fed corn silage-based diets containing 164 g/kg of crude protein and 6.72 MJ/kg of net energy. Eight rumen cannulated Holstein cows (207 ± 23.6 days in milk and 33.4 ± 2.45 kg/d of milk yield) were used in a 4 × 4 Latin square design with a 2 × 2 factorial treatment structure. Treatments consisted of diets with or without CSFA at 33.2 g/kg dry matter (DM) combined with either low (98 g/kg DM) or high level (110 g/kg DM) of RDP. Microbial protein synthesis was evaluated using the omasal sampling technique along with the triple marker system of digesta flow and 15N as a microbial marker. There was no interaction effect (P ≥ 0.15) between CSFA and RDP for all variables analyzed. Fat supplementation reduced (P ≤ 0.010) intake of nutrients (except ether extract), tended to increase (P = 0.076) rumination time, and increased (P = 0.015) ruminal pH. Calcium salts of fatty acids decreased (P ≤ 0.014) DM and organic matter ruminal digestibility and tended to reduce (P = 0.096) neutral detergent fiber ruminal digestibility. Dietary CSFA did not affect (P ≥ 0.32) ruminal flows of total non-ammonia N and microbial non-ammonia N, but increased (P = 0.022) microbial protein synthesis efficiency. Cows fed CSFA had greater (P ≤ 0.043) milk yield, feed efficiency, and N secreted in milk (g/kg N intake), and lower (P < 0.001) milk fat content in comparison with counterparts. Cows fed the diets with low RDP tended to have greater (P = 0.096) crude protein (CP) intake in comparison with cows fed diets with high RDP. Cows fed low RDP tended to exhibit greater (P = 0.057) ruminal acetate to propionate ratio than those fed high RDP. Low RDP decreased (P ≤ 0.048) starch total tract digestibility and ruminal protein degradation, tended to increase (P = 0.055) milk yield, and reduced (P = 0.002) milk protein content, but did not affect (P = 0.76) microbial protein synthesis efficiency compared to high RDP. Thus, lower RDP increased non-microbial non-ammonia nitrogen and reduced urinary losses of N, whereas CSFA decreased feed intake and improved microbial protein synthesis efficiency in mid-lactating cows. However, RDP level and CSFA association shows no interaction effect.