AbstractHIV‐1 Nef accelerates the progression to AIDS by binding with and activating a Src kinase Hck, but underlying molecular basis is not understood. We revealed that Nef disturbed N‐glycosylation/trafficking of a cytokine receptor Fms in an Hck‐dependent manner, a possible trigger to worsen uncontrolled immune system. Here, we provide direct evidence that dys‐regulated activation of Hck pre‐localized to the Golgi apparatus causes this Fms maturation arrest. A striking change in Hck induced by Nef other than activation was its skewed localization to the Golgi due to predominant Golgi‐localization of Nef. Studies with different Nef alleles and their mutants showed a clear correlation among higher Nef‐Hck affinity, stronger Hck activation, severe Golgi‐localization of Hck and severe Fms maturation arrest. Studies with a newly discovered Nef‐Hck binding blocker 2c more clearly showed that skewed Golgi‐localization of active Hck was indeed the cause of Fms maturation arrest. 2c blocked Nef‐induced skewed Golgi‐localization of an active form of Hck (Hck‐P2A) and Fms maturation arrest by Nef/Hck‐P2A, but showed no inhibition on Hck‐P2A kinase activity. Our finding establishes an intriguing link between the pathogenesis of Nef and a newly emerging concept that the Golgi‐localized Src kinases regulate the Golgi function. J. Cell. Physiol. 221: 458–468, 2009. © 2009 Wiley‐Liss, Inc.