To minimize dependency on chemical pesticides, plant breeders are trying to emphasize on important agricultural pests for the development of pest resistant cultivars. However, the molecular approach and associated genetic tools conferring resistance have not been widely studied. In the current study, proteomic analysis of two of the alfalfa cultivars viz. a resistant (R) (Zhongmu-1) and a susceptible (S) (WL343), with (+ A) and without (− A) aphids rearing were carried out. Results indicated that 325 differentially expressed proteins (DEPs) up-regulated while 319 down-regulated with a pattern of R + A/R − A plants, whereas 371 up-and 583 down-regulated DEPs were identified in the S + A/S − A plants. Total number of DEPs found in (S + A/S -A) was around 19.7% greater than that of (R + A/R -A), whereas, the down-regulated DEPs of susceptible variety was 11.6% higher than the resistant cultivar. Applying the KEGG analysis, 96 and 142 DEPs were portrayed to 15 and 10 substantively augmented pathways for Zhongmu-1 and WL343, respectively. We also found that two of the shared pathways (carbon metabolism and pyruvate metabolism) are linking to important traits conferring resistance in alfalfa. Most importantly, the specific role of linoleic acid metabolism was found to be associated with jasmonic acid, flavonoid biosynthesis, and terpenoid backbone biosynthesis that might have been associated with the insect-resistant material synthesis in the resistant alfalfa cultivar. Our study suggested that both alfalfa cultivars (R, S) could govern protein expression through carbon and pyruvate metabolism. But only the resistant alfalfa cultivar (Zhongmu-1) can tune protein expression via linoleic acid metabolism and terpenoid backbone biosynthesis to induce the defensive protein expressions (e.g., jasmonic acid and flavonoid biosynthesis along with terpenoid backbone biosynthesis), to enhance plant defense capacity.