Dengue, caused by the dengue virus (DENV), presents a significant public health challenge with limited effective treatments. NITD-688 is a potent pan-serotype DENV inhibitor currently in Phase II clinical trials. However, its mechanism of action is not fully understood. Here, we present the molecular details of how NITD-688 inhibits DENV. NITD-688 binds directly to nonstructural protein 4B (NS4B) with nanomolar affinities across all four DENV serotypes. This binding specifically disrupts the interaction between NS4B and nonstructural protein 3 (NS3), without affecting interactions with other viral or host proteins. NITD-688-resistant mutations in NS4B reduce NITD-688 binding to NS4B and its disruption of the NS4B/NS3 interaction. Mechanistically, NITD-688 blocks the interaction of NS3 with a cytosolic loop within NS4B and disrupts both the formation and pre-existing NS4B/NS3 complexes in vitro and in DENV-infected cells, ultimately inhibiting viral replication. This characteristic enables NITD-688 to remain effective even with delayed treatment, distinguishing it from JNJ-1802, another clinically studied NS4B inhibitor. Moreover, combining NITD-688 with either JNJ-1802 or an NS5 polymerase inhibitor synergistically inhibits DENV-2 infection. Together, these findings provide critical insights into the mechanism of action of NITD-688, facilitating the development of novel flavivirus NS4B inhibitors and informing future clinical interventions against DENV.