Infectious bursal disease virus (IBDV) causes an acute, highly contagious immunosuppressive disease—infectious bursal disease (IBD) in poultry. It is known that VP4 is a non-structural protein and a viral protease encoded by IBDV. Currently, little is known about VP4 characteristics during pathogenic and nonpathogenic IBDV infection. Here, we investigated the expression profiles of VP4 during pathogenic and nonpathogenic IBDV infection. By IFA and ELISA, using VP4 protein respectively expressed in Vero cells transfected with VP4 gene and in Escherichia coli as antigens, we firstly confirmed serum anti-VP4 antibodies in pathogenic IBDV-infected rather than nonpathogenic IBDV-infected chickens. Kinetic analysis of anti-IBDV antibody shows that in the pathogenic IBDV-infected chickens, the antibody to VP4 was later detectable than anti-VP3 antibody and virus neutralizing antibody. Immunohistochemistry further demonstrates that VP4 antigen can be detected mainly in the cortex of lymphoid follicles of bursa of Fabricius infected with pathogenic IBDV. These data first suggest that VP4 antibody is an indicator discriminating pathogenic and nonpathogenic IBDV infection in chickens.