Maize rough dwarf disease (MRDD) is a severe disease that has been occurring frequently in southern China and many other Asian countries. MRDD is caused by the infection of Rice black streaked dwarf virus (RBSDV) and leads to significant economic losses in maize production. To well understand the destructive effects of RBSDV infection on maize growth, comparative proteomic analysis of maize seedlings under RBSDV infection was performed using an integrated approach involving LC-MS/MS and TMT labeling. Our study identified 7615 maize proteins, of which 6319 proteins were quantified. A total of 116 differentially expressed proteins (DEPs) were identified, including 35 up- and 81 down-regulated proteins under RBSDV infection. Enrichment analysis showed that the DEPs were most strongly associated with Cyanoamino acid metabolism, protein processing in ER, and ribosome-related pathways. Two sulfur metabolism-related proteins were significantly reduced, indicating that sulfur may participate in the resistance against RBSDV infection. Furthermore, 15 DEPs involved in six metabolic pathways were identified in maize under RBSDV infection. Our data revealed that the responses of maize to RBSDV infection were controlled by various metabolic pathways.