Dengue virus (DENV) is the important causative agent of dengue hemorrhagic fever and dengue shock syndrome that are potentially life-threatening diseases affecting human population globally. At present, the underlying mechanism of dengue diseases remains unclear and no dengue vaccine is commercially available. One of the crucial viral products conceivably involved in DENV production and dengue pathogenesis is the nonstructural protein 1 (NS1). The NS1 protein expresses in multiple forms in different cellular compartments with possibly distinct functions. This study was carried out to identify host cellular proteins interacting with cell-associated NS1 in both DENV infection and NS1 transfection systems and to investigate their interactive roles in DENV life cycle and host response against DENV infection by using biochemical and proteomic approaches. As evidenced by liquid chromatography tandem-mass spectrometry (LC MS/MS), two separate groups of novel host cellular proteins have been identified from DENV-infected HepG2 cells and NS1-expresing Huh7 cells to be potential NS1-interacting partners. Some of the selected proteins have been confirmed for their association with DENV NS1 in virus-infected cells by co-immunoprecipitation assay. Additionally, hnRNP C1/C2 were found to interact with DENV NS1 and viral RNA and moderately influence DENV life cycle at the steps of viral RNA replication and viral protein synthesis. GRP78 was also another host cellular protein interacting with DENV NS1 and likely played a role in viral protein processing. Taken together, our study provided new evidence for potential DENV NS1-inteacting host cellular proteins in DENV-infected hepatocyte cells and addressed certain aspects of host protein and DENV NS1 association in DENV life cycle. Further investigation on detailed molecular mechanisms required for their specific interactions would be needed to pave the way for future research on anti-viral drug development.