Astrocytes are key regulators of central nervous system (CNS) health and neuronal function. Astrocyte mitochondrial dysfunction, such as induced by METH and HIV-1, threatens the provision of essential metabolic and antioxidant support to neurons. Thus, delineating regulatory pathways that can be targeted to prevent aberrant mitochondria homeostasis in astrocytes will be imperative for ensuring neuronal fitness/survival against CNS pathologies. Direct contact sites between the endoplasmic reticulum (ER) and the mitochondria, termed mitochondrial associated membranes (MAMs), are central hubs for regulating several cellular processes required for homeostasis, including mitochondrial metabolic activity. In fact, the transfer of Ca2+ from the ER to mitochondria is essential for mitochondrial bioenergetics. Recent investigations have also identified unique, yet ill-defined contributions of the three unfolded protein response (UPR) arms, beyond their classical ER stress functions, in regulating MAM tethering and/or signaling. Briefly, protein kinase RNA-like endoplasmic reticulum kinase (PERK) has been determined as a key regulator for MAM tethering, inositol-requiring kinase 1 (IRE1α) is implicated in regulating MAM-mediated Ca2+ transfer, and activating transcription factor 6 (ATF6) is suspected to participate in MAM formation as it is known to mediate ER elongation and lipid homeostasis. However, these regulatory mechanisms have not yet been fully elucidated. The current investigation examined changes in astrocyte mitochondrial function, ER stress, Ca2+ signaling, and the regulation of MAMs in response to METH exposure and HIV-1 infection. Then, we explored the role of ER-associated mechanisms in regulating astrocyte mitochondrial function. The effects of METH treatment were evaluated in both acute and chronic paradigms while responses to chronic HIV-1 infection was examined using a pseudotyped HIV-1 virion able to infect astrocytes. Astrocyte metabolic status was determined using Seahorse extracellular flux analyzer for a real-time assessment of cellular metabolism in addition to quantifying the expression profiles of essential metabolites. Changes in the expression of unfolded protein response (UPR) and MAM mediators were determined using RT-PCR and protein expression assays. Finally, pharmacological inhibition of the UPR pathways were used to delineate the ER-associated regulatory mechanisms mediating the changes in mitochondria bioenergetics. Our studies demonstrate increased astrocyte metabolic capacity in response to chronic METH exposure and HIV-1 infection which corresponded to increased expression of UPR/MAM mediators. Moreover, pharmacological inhibition of specifically IRE1α impaired astrocyte mitochondrial activity. These findings illustrate the importance of ER-mitochondria communication in regulating astrocyte mitochondrial function and identifies a possible mechanism to manipulate the metabolic and antioxidant coupling between astrocytes and neurons during METH/HIV-1 pathogenesis.