Arginine is classified as a conditionally essential amino acid required exogenously during catabolic disease states and periods of rapid growth, both characterized by increased arginine utilization. Arginine plays an important role in the intestine, where it is extensively metabolized, and enhances its immune-supportive function and mucosal repair. Cell proliferation is important for the latter process. This study aimed for a better molecular insight in the response to arginine deprivation/supplementation of preconfluent and 5-day-confluent, differentiated Caco-2 intestinal cells. The potential of citrulline to counteract the effects of arginine deprivation was investigated in preconfluent cells. 2-DE combined with MALDI-TOF-MS and the antibody microarray technology were applied. Evidence is provided that arginine deficiency modulates the protein expression profiles of preconfluent Caco-2 cells differently than that of postconfluent differentiated cells. In preconfluent cells, certain proteins changed in direct response to arginine deficiency, whereas other proteins did not, but instead responded during the recovery phase after an arginine/citrulline resupplementation. The protein changes suggest that arginine deprivation decreases cell proliferation and heat shock protein expression, and enhances the cells susceptibility to apoptosis. These processes are critical for proper cell function, and hence a state of arginine deficiency can be detrimental for intestinal cells which proliferate actively in vivo.