During translation of the flavivirus polyprotein, prM is the first protein translocated into the ER lumen, where it acts as a chaperone during the folding of the subsequently translocated E protein [4, 37, 44] . In addition to this important role of prM during E protein synthesis, a variety of data suggest that the interaction of pr peptide with the viral E protein protects flaviviruses from low pH during their transport through the exocytic pathway [34, 35, 36] . Here we showed that a recombinant pr peptide was efficiently folded, glycosylated, and secreted from 293T cells in the absence of its normal prM context and furin processing. Recombinant pr bound to soluble E proteins at low pH, inhibited E-membrane insertion, and interacted with mature dengue virus to block fusion and infection. Alanine substitution of the conserved E H244 within the pr-E interface disrupted pr-E binding in vitro and blocked secondary virus infection. VLP production was inhibited by the H244A mutation and partially rescued by pH neutralization with NH 4 Cl. Together our data demonstrate the critical role of pr in protecting DENV from exocytic low pH.