Infantile spasms is an age-specific epileptic syndrome associated with poor developmental outcomes and poor response to nearly all traditional antiepileptic drugs except adrenocorticotropic hormone (ACTH). We investigated the protective mechanism of ACTH against brain damage. An infantile spasm rat model induced by N-methyl-D-aspartate (NMDA) in neonate rats was used. Pregnant rats were randomly divided into the stress-exposed and the non-stress exposed groups, and their offspring were randomly divided into ACTH-treated spasm model, untreated spasm model, and control groups. A proteomics-based approach was used to detect the proteome differences between ACTH-treated and untreated groups. Gel image analysis was followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric protein identification and bioinformatics analysis. Prenatal stress exposure resulted in more severe seizures, and ACTH treatment reduced and delayed the onset of seizures. The most significantly up-regulated proteins included isoform 1 of tubulin β-5 chain, cofilin-1 (CFL1), synaptosomal-associated protein 25, malate dehydrogenase, N(G),N(G)-dimethylarginine dimethylaminohydrolase 1, annexin A3 (ANXA3), and rho GDP-dissociation inhibitor 1 (ARHGDIA). In contrast, tubulin α-1A chain was down-regulated. Three of the identified proteins, ARHGDIA, ANXA3, and CFL1, were validated using western blot analysis. ARHGDIA expression was assayed in the brain samples of five infantile spasm patients. These proteins are involved in the cytoskeleton, synapses, energy metabolism, vascular regulation, signal transduction, and acetylation. The mechanism underlying the effects of ACTH involves the molecular events affected by these proteins, and protein acetylation is the mechanism of action of the drug treatment.