Rotaviruses (RVs) are a leading cause of acute viral gastroenteritis in young children and livestock worldwide. Growing evidence suggests that host cellular glycans, such as histo-blood group antigens (HBGAs) and sialic acids (SA), are recognized by the RV surface protein VP4. However, a mechanistic understanding of these interactions and their effects on RV infection and pathogenesis is lacking. Here, we established a porcine crypt-derived 3D intestinal enteroids (PIEs) culture system which contains all intestinal epithelial cells identified in vivo and represents a unique physiologically functional model to study RV-glycan interactions in vitro. PIEs expressing different HBGAs (A+, H+, and A+/H+) were established and isolation, propagation, differentiation and RV infection conditions were optimized. Differentiated PIEs were infected with human RV (HRV) G1P[8] Wa, porcine RV (PRV) G9P[13], PRV Gottfried G4P[6] or PRV OSU G5P[7] virulent and attenuated strains and virus replication was measured by qRT-PCR. Our results indicated that virulent HRV G1P[8] Wa replicated to the highest titers in A+ PIEs, while a distinct trend was observed for PRV G9P[13] or G5P[7] with highest titers in H+ PIEs. Attenuated Wa and Gottfried strains replicated poorly in PIEs while the replication of attenuated G9P[13] and OSU strains in PIEs was relatively efficient. However, the replication of all 4 attenuate strains was less affected by the PIE HBGA phenotypes. HBGA synthesis inhibitor 2-F-Peracetyl-Fucose (2F) treatment demonstrated that HBGAs are essential for G1P[8] Wa replication; however, they may only serve as a cofactor for PRVs G9P[13] and OSU G5P[7]. Interestingly, contrasting outcomes were observed following sialidase treatment which significantly enhanced G9P[13] replication, but inhibited the growth of G5P[7]. These observations suggest that some additional receptors recognized by G9P[13] become unmasked after removal of terminal SA. Overall, our results confirm that differential HBGAs-RV and SA-RV interactions determine replication efficacy of virulent group A RVs in PIEs. Consequently, targeting individual glycans for development of therapeutics may not yield uniform results for various RV strains.