Because of their similarities to infants in mucosal immune responses and their susceptibility to human rotavirus (HRV) diarrhea, gnotobiotic pigs provide a useful model for rotaviral disease. In this study, we performed quantitative enzyme-linked immunospot (ELISPOT) assays to measure local and systemic isotype-specific antibody-secreting cell (ASC) responses to individual structural (VP4, VP6, and VP7) and nonstructural (NSP3 and NSP4) proteins of Wa HRV. The Spodoptera frugiperda cells expressing each recombinant baculovirus HRV protein were formalin fixed and used as antigen for ELISPOT assays. Neonatal gnotobiotic pigs were orally inoculated once with virulent Wa (WaV) or three times with attenuated Wa (WaA) HRV or mock inoculated (Mock) and then were challenged with virulent Wa (WaV/PC) 28 days after the first inoculation. The ASCs from intestinal and systemic lymphoid tissues of pigs from each group were quantitated by ELISPOT assay at the day of challenge, at postinoculation day 28 (WaV, WaA, and Mock) or at postchallenge day (PCD) 7 (WaV+WaV/PC, WaA+WaV/PC, and Mock+WaV/PC). In all virus-inoculated pigs, regardless of the inoculum, lymphoid tissue, or isotype, VP6 induced the highest numbers of ASCs, followed by VP4; ASCs specific for VP7, NSP3, and NSP4 were less numerous. At challenge, total HRV- and HRV protein-specific immunoglobulin A (IgA) and IgG ASCs in intestinal lymphoid tissues were significantly greater in WaV- than in WaA-inoculated pigs, and WaV pigs were fully protected against diarrhea postchallenge, whereas the WaA pigs were partially protected. At PCD 7, there were no significant differences in ASC numbers for any HRV proteins between the WaV+WaV/PC and WaA+WaV/PC groups.