Human immunodeficiency virus (HIV) infections remain a significant public health burden globally with infected individuals at high risk for cognitive decline and memory loss even on combination antiretroviral therapy. Almost half of HIV infected individuals smoke, which drives poorer health outcomes including a higher dementia rate. Microglia are the brain's immune cells that serve as a persistent HIV reservoir contributing to neuroinflammatory signaling. We examined interactions between the HIV envelope glycoprotein gp120 and nicotine within human microglia cells (HMC3) that endogenously express chemokine receptor 5 (CCR5) and nicotinic acetylcholine receptors (nAChRs). Liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI/MS) shows that gp120 alters mitochondria proteins within HMC3 cells. In the presence of nicotine, gp120 increased the expression of mitochondrial prohibitin 2 (PHB2), cytochrome c (cyt c), and mitofusin 2 (MFN2) but decreased fission 1 (FIS1) levels. An analysis of mito-YFP expression confirms that interaction between nicotine and gp120 increases the size and branching of mitochondrial networks. Interaction between nicotine and gp120 is also surprisingly found to promote the release of amyloid precursor protein (APP) peptides from microglia. This was accompanied by visualization of amyloid containing vesicles that colocalized with the autophagy protein LC3B-II in the cell. Taken together, our findings show that interaction between nicotine and gp120 impact microglia in a manner that regulates mitochondrial proteins and network properties and impacts amyloid protein management and release within microglia. These mechanisms may contribute to understanding neuroinflammatory signaling in smokers with HIV.