Two newly developed transgenic alfalfa with transformed TRANSPARENT TESTA 8 (TT8) and HB12 genes were developed by scientists at Agriculture and Agri-Food Canada. Our study was conducted to (1) predict the extent of ruminal and intestinal digestion of protein, (2) evaluate the nutrient supply to dairy cattle and (3) determine the feed milk value in dairy cattle. HB12 transgenic alfalfa, TT8 transgenic alfalfa and unmodified control genotype alfalfa were cultivated and harvested from the greenhouse of Agriculture and Agri-Food Canada. There were 2 experimental samples for each treatment. Total rumen degraded protein (TRDP), total rumen undegraded protein (TRUP) and intestinal digestible protein fractions were predicted. The parameters evaluated included: truly absorbed rumen microbial protein (AMCP), truly absorbed rumen undegraded feed protein in the small intestine (ARUP), endogenous protein loss in the small intestine (ENDP), total metabolizable protein (DVE) and degraded protein balance (OEB). The PROC MIXED procedure of SAS 9.3 was used for data analysis. Significance was declared at P < 0.05 and trends at P ≤ 0.10. The results showed that TT8 alfalfa had highest TRDP value compared with HB12 and control alfalfa (P < 0.05). TT8 alfalfa trended to be higher in TRUP than HB12 alfalfa, with control alfalfa intermediate (P < 0.10). Intestinal digestible PB1 was higher in TT8 alfalfa than the other 2 alfalfa plants (P < 0.05). TT8 alfalfa had a greater content of microbial protein production (MCP_RDP, P < 0.05). No significant difference of OEB value was found between HB12 alfalfa and control alfalfa, but the OEB value was higher in TT8 alfalfa (P < 0.05). No significant differences in DVE and feed milk value (FMV) were found among 3 different genotypes of alfalfa plants (P > 0.05). In conclusion, altering the expression of lignin-related TT8 and HB12 genes in alfalfa plant could affect its nutrient supply to dairy cattle.