Background HIV‐1 Nef regulates several cellular functions in an infected cell which results in viral persistence and AIDS pathogenesis. The currently understood molecular mechanism(s) underlying Nef‐dependent cellular function(s) are unable to explain how events are coordinately regulated in the host cell. Intracellular membranous trafficking maintains cellular homeostasis and is regulated by Rab GTPases ‐ a member of the Ras superfamily. Results In the current study, we tried to decipher the role of Nef on the Rab GTPases‐dependent complex and vesicular trafficking. Expression profiling of Rabs in Nef‐expressing cells showed that Nef differentially regulates the expression of individual Rabs in a cell‐specific manner. Further analysis of Rabs in HIV‐1NL4‐3 or ΔNef infected cells demonstrated that the Nef protein is responsible for variation in Rabs expression. Using a panel of competitive peptide inhibitors against Nef, we identified the critical domain of HIV‐1 Nef involved in modulation of Rabs expression. The molecular function of Nef‐mediated upregulation of Rab5 and Rab7 and downregulation of Rab11 increased the transport of SERINC5 from the cell surface to the lysosomal compartment. Moreover, the Nef‐dependent increase in Rab27 expression assists exosome release. Reversal of Rabs expression using competitive inhibitors against Nef and manipulation of Rabs expression reduced viral release and infectivity of progeny virions. Conclusion This study demonstrates that Nef differentially regulates the expression of Rab proteins in HIV‐1 infected cells to hijack the host intracellular trafficking, which augments viral replication and HIV‐1 pathogenesis. Significance Our study emphasized the indispensable role of HIV‐1 protein Nef on various aspects of the intracellular trafficking regulated by Rabs GTPases, which explained how HIV‐1 Nef may hijack membrane trafficking pathways in infected cells. HIV‐1 Nef hijacks the host intracellular trafficking pathways by manipulating the expression of Rab GTPases in a‐cell specific manner. Nef‐dependent differential regulation of Rab5, Rab11, and Rab7 accelerate endo‐lysosomal trafficking of viral restriction factor SERINC5 and Rab27 upregulation promotes release of exosomes. Reversal of Nef‐induced differential regulation of Rabs expression by competitive inhibitors reduces viral release and infectivity of progeny virions which could be considered as an anti‐retroviral therapeutic agent.