The nucleoside reverse transcriptase inhibitors (NRTIs), used for treatment of the human immunodeficiency virus-1, compromise mitochondria in cardiomyocytes and other host cells, limiting the clinical use of these drugs. To explore underlying mechanisms, we overexpressed PGC-1α, a master regulator of mitochondrial biogenesis, twofold in H9c2 rat cardiomyocyte cultures, hypothesizing that this might protect the mitochondria from damage induced by the NRTI combination zidovudine (AZT) and didanosine (ddI). The experimental groups, evaluated during 16 passages (P) of drug exposure, included: PGC-1α-overexpressing cells with no exposure, or exposure to 50 µM AZT plus 50 µM ddI; and control cells with no exposure or exposure to the same doses of AZT and ddI. The AZT/ddI combination caused a growth inhibition of 15-20% in control cells, but none in PGC-1α cells. Apoptosis was highest in AZT/ddI-exposed control cells, and PGC-1α overexpression protected cells from AZT/ddI-induced apoptosis. At P3, P6, P8, and P12, uncoupled mitochondrial oxygen consumption rate, determined by Seahorse 24 XF Analyzer, as higher in AZT/ddI-exposed PGC-1α cells, compared to AZT/ddI-exposed control cells (p < 0.05 at all P). Complex I activity was higher in AZT/ddI-exposed PGC-1α overexpressing cells than that in AZT/ddI-exposed control cells (p < 0.05), and reactive oxygen species levels were lower in PGC-1α overexpressing cells than that in control cells (p < 0.05) when both were exposed to AZT/ddI. Taken together, these experiments show proof of concept that overexpression of PGC-1α protects cardiomyocytes from NRTI-induced toxicity, and suggest that a pharmaceutical agent with similar activity may protect against NRTI-induced mitochondrial toxicity.