Eyestalk ablation is a common technique to induce spawning in marine shrimps. Currently, little is known about how this technique affects nutritional preferences (energy sources) in response to stress induced by eyestalk ablation during reproduction and growth. Thus, this study investigated the physiological effects of eyestalk ablation on hemolymph nutrient (glucose and protein) utilization and plasma protein expression of the female giant freshwater prawn (Macrobrachium rosenbergii). Sixty female M. rosenbergii prawns weighing 15.6 ± 0.3 g were divided into unablated (no eyestalk ablation, n = 30) and ablated (unilateral eyestalk ablation, n = 30) groups. The groups were then allowed to undergo reproductive and nonreproductive molting cycles. During normal molting, hemolymph protein (1,339.90 ± 95.08 mg/mL) concentrations decreased in the postmolt stage during the reproductive cycle, indicating that protein was used to attain full ovarian maturation, as evidenced by a large rise in the Gonadosomatic index value (8.47% ± 0.52%). The ablated group was vulnerable to stress, especially during the premolt (reproductive) stage, as indicated by a significant (P < 0.05) reduction in hemolymph protein concentrations, whereas no such stress-related effect was observed during the premolt (nonreproductive) stage. Interestingly, protein profiles were unaffected by eyestalk ablation, as shown by the similarity in profiles in both the unablated and ablated groups. Using a proteomic approach, fragment hemocyanin subunit 2, an immune-related protein, was identified in the ablated group. Overall, the results suggested that protein appeared to be the main nutritional source for reproduction and growth during short-term stress associated with eyestalk ablation, which led to stimulation of hemocyanin subunit 2.