Central nervous system disorders are still a common complication of HIV infection and can lead to dementia and death. They are mostly the consequences of an inflammatory macrophagic activation and of glutamate-mediated excitotoxicity. However, recent studies also suggest neuroprotective aspects of macrophage activation through the expression of glutamate transporters and glutamine synthetase. We thus aimed to study whether HIV infection or activation of macrophages could modulate glutamate metabolism in these cells. We assessed the effect of HIV infection on glutamate transporter expression as well as on glutamate uptake by macrophages and showed that glutamate transport was partially decreased in the course of virus replication whereas EAAT-2 gene expression was dramatically increased. The consequences of HIV infection on glutamine synthetase was also measured and for the first time we show the functional expression of this key enzyme in macrophages. This expression was repressed during virus production. We then quantified EAAT-1 and EAAT-2 gene expression as well as glutamate uptake in differentially activated macrophages and show that the effects of HIV are not related to pro- or anti-inflammatory mediators. Finally, this study shows that the glutamate transport by macrophages is less affected by HIV infection than what was described in astrocytes. Macrophages may thus play a role in neuroprotection against glutamate in the infected brain, through their expression of both EAATs and glutamine synthetase. As glutamate metabolism by activated macrophages is sensitive to both HIV infection and inflammation, it may thus be of potential interest as a therapeutic target in HIV encephalitis.