Chikungunya is a febrile infection caused by the Chikungunya virus (CHIKV), an alphavirus which has emerged as a serious public health problem globally. Despite extensive research, our understanding of different host factors facilitating effective CHIKV infection is not clear yet. NEDD4, a member of the E3 ubiquitin ligase, is one such protein. Here, the importance of NEDD4 has been explored during CHIKV infection in vitro. It was observed that the level of NEDD4 is downregulated after CHIKV infection. Interestingly, the CHIKV-nsP3 level and the viral load were decreased significantly when NEDD4 was silenced, while a 93% decrease in the viral load was observed in the case of NEDD4 overexpression, indicating the importance of an optimum level of NEDD4 for effective CHIKV infection. Further study revealed that there was interaction between the NEDD4 and CHIKV-nsP3 proteins through co-immunoprecipitation (CO-IP) during CHIKV infection. Additionally, in silico data illustrated that the WW domain of NEDD4 can bind to the nsP3, as well as the macrodomain of nsP3 (nsp3-MD) of CHIKV. These data were further confirmed by the pull-down assay with purified nsP3-MD. The finding suggested that the host protein NEDD4 might interact directly with nsP3-MD during the CHIKV infection. However, the presence of a faint band of NEDD4 along with nsP3-MD in the pull-down assay may indicate the involvement of some other residues for this interaction. These in silico data were further confirmed by the CO-IP experiments, where all domains of nsP3, MD (macrodomain), AUD (alphavirus unique domain) and HVD (hypervariable domain) were found to interact with NEDD4. Additional experiments with a truncated form of MD, MD1 (1–100 residues of amino acid), revealed that this region is not able to maintain the interaction with NEDD4, indicating the crucial role of the C-terminal region of MD for this binding. In conclusion, these findings offer valuable insights about the importance of NEDD4 during CHIKV infection and the residues of nsP3 for its interaction, which might be useful to design future therapeutics against CHIKV.