Use of canola meal (CM) and dried corn distillers grains with solubles (DDGS) as major supplemental protein sources are common practice in North American dairy rations and usage of both is projected to increase in the future. Since limited data is available on performance of cows fed diets with different ratios of CM and DDGS, our objective was to determine the optimal ratio of CM to DDGS protein in a contemporary lactation dairy ration by feeding combinations of CM and high protein DDG (HPDDG) to early lactation multiparity dairy cows. The experiment was a 4×4 Latin square with 28 d periods using four pens of ∼320 high producing cows/pen. Treatments were created by varying the amounts of CM and HPDDG added on a DM basis to be: (1) 0 g/kg CM and 200 g/kg HPDDG, (2) 65 g/kg CM and 135 g/kg HPDDG, (3) 135 g/kg CM and 65 g/kg HPDDG, (4) 200 g/kg CM and 0 g/kg HPDDG. Dry matter intake was not affected by the CM/HPDDG ratio in the ration. Milk and lactose yield, true protein (TP) content and yield, milk fat yield as well as milk energy output increased at a decreasing rate with a higher CM/HPDDG ratio. Maximum values for milk and TP yield were at ∼135 g/kg CM, while lactose, TP content and milk energy were maximized at ∼120 g/kg CM inclusion. Milk fat content and milk energy density decreased linearly with higher CM inclusion. Body condition score change responded quadratically with the highest gain at ∼120 g/kg CM inclusion. The purine derivative to creatinine index increased linearly with higher CM inclusion levels, suggesting that microbial protein production (MCP) was limited in the 0 g/kg CM ration and was progressively stimulated by higher feeding levels of CM. Plasma AA levels suggest that the reduction in lysine in dietary protein, together with the decrease in MCP production, resulted in a substantial reduction in lysine available for milk production, thereby limiting performance in the higher HPDDG ration. The only AA which decreased in plasma with higher CM feeding levels were phenylalanine, leucine and methionine. That the level of leucine in the plasma was still decreasing linearly, while methionine and phenylalanine responded quadratically at the 200 g/kg CM treatment, was interpreted to suggest that the leucine supply remained higher than its requirement at the highest CM inclusion level, but that phenylalanine and/or methionine was limiting production in the highest CM ration. Overall, results suggest that the optimum ratio of CM to HPDDG in these diets was with 120–135 g/kg of diet DM from CM.