Although alterations in mitochondrial dynamics are associated with cellular responses to injury, the functional role of these dynamic changes in ischemic neurons is underexplored. One of these dynamic responses to injury includes mitochondrial biogenesis. Various sublethal pre‐conditioning stimuli that induce an ischemic‐tolerant state [e.g., lipopolysaccharide (LPS)] may also induce mitochondrial biogenesis. Using neuron‐enriched cultures, we found that sublethal LPS pre‐conditioning induced both ischemic tolerance and markers of mitochondrial biogenesis with overlapping dose–response temporal kinetics. Sublethal LPS transiently increased the expression of critical components of the mitochondrial transcriptional machinery, including nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM), as well as mtDNA copy number, mitochondrial protein levels, and markers of functional mitochondria, such as increased cellular ATP content, citrate synthase activity, and maximal respiration capacity. Importantly, knockdown of TFAM abrogated both the induction of mitochondrial biogenesis and the neuroprotective pre‐conditioning effects of LPS. Several signaling pathways coordinated these events. AMPK inhibition suppressed NRF1 and TFAM expression by LPS, whereas PI3K/Akt signaling was necessary for the nuclear translocation of NRF1 and subsequent induction of TFAM. This is the first demonstration that LPS pre‐conditioning initiates multiple signaling pathways leading to mitochondrial biogenesis in neurons and that these dynamic changes contribute to ischemic tolerance.