The interaction between HIV-1 Nef and the Src kinase Hck in macrophages has been shown to accelerate the progression to AIDS. We previously showed that Nef disturbed the N-glycosylation/trafficking of Fms, a cytokine receptor essential for maintaining macrophages in an anti-inflammatory state, in an Hck-dependent manner. Here, we show the underlying molecular mechanism of this effect. Using various Hck isoforms and their mutants and Golgi-targeting Hck mutants, we confirmed that Hck activation at the Golgi causes the Nef-induced Fms N-glycosylation defect. Importantly, we found that both the co-expression of Nef and Hck and the expression of a Golgi-targeted active Hck mutant caused alterations in the distribution of GM130, a Golgi protein that was shown to be required for efficient protein glycosylation. Moreover, the activation of Hck at the Golgi caused strong serine phosphorylation of the GM130-interacting Golgi structural protein GRASP65, which is known to induce Golgi cisternal unstacking. Using pharmacological inhibitors, we also found that the activation of Hck at the Golgi followed by the activation of the MAP kinase ERK-GRASP65 cascade is involved in the Fms N-glycosylation defect. These results suggest that Nef perturbs the structure and signaling of the Golgi by activating Hck at the Golgi, and thereby, induces the N-glycosylation/trafficking defect of Fms, which is in line with the idea that Src family kinases are crucial Golgi regulators. J. Cell. Physiol. 227: 1090–1097, 2012. © 2011 Wiley Periodicals, Inc.