Infectious entry of the nonenveloped rotavirus virion requires proteolysis of the spike protein VP4 to mediate conformational changes associated with membrane penetration. We sequenced and characterized an isolate that was cultured in the absence of trypsin and found that it is more resistant to proteolysis than WT virus. A substitution mutation abrogates one of the defined trypsin-cleavage sites, suggesting that blocking proteolysis at this site reduces the overall kinetics of proteolysis. Kinetic analysis of the membrane penetration-associated conformational change indicated that the 'fold-back' of the mutant spike protein is slower than that of WT. Despite these apparent biochemical defects, the mutant virus replicates in an identical manner to the WT virus. These findings enhance an understanding of VP4 functions and establish new strategies to interrogate rotavirus cell entry.