Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.Group A rotavirus (RV) is the leading cause of acute gastroenteritis in human infants less than 5 years old, causing 611,000 deaths per year (41). It is also the main cause of severe diarrhea in the neonates of many animal species of economic interest (43,47). RV virions are triple-layered particles composed by a core (protein VP2), an inner capsid (protein VP6), and an outer capsid (proteins VP7 and VP4) (16,29). The inner capsid protein, VP6, is a trimer representing 51% of the virion mass. According to the antigenic variation of VP6, RVs are classified into seven groups (A to G) (16). Depending on the presence or absence of two different epitopes in the VP6 protein, group A RV strains are further divided into subgroups (Sb) I, II, IĎ©II, and no I no II. Despite the different subgroups mentioned, VP6 is a strongly conserved protein among all group A RVs (Ďľ90% amino acid homology). It is highly immunogenic and constitutes the target antigen of most immunodiagnosis tests for group A RV detection. In contrast, the outer capsid proteins VP7 (glycoprotein) and VP4 (protease sensitive) are highly variable and constitute the major neutralizing antigens. Based on the variation of VP7 and VP4, group A RVs are further classified into G and P types, respectively.