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<i>Toxoplasma gondii</i> (<i>T. gondii</i>) is an opportunistic protozoan that can cause brain infection and other serious health consequences in immuno-compromised individuals. This parasite has a remarkable ability to cross biological barriers and exploit the host cell microenvironment to support its own survival and growth. Recent advances in label-free spectroscopic imaging techniques have made it possible to study biological systems at a high spatial resolution. In this study, we used conventional Fourier-transform infrared (FTIR) microspectroscopy and synchrotron-based FTIR microspectroscopy to analyze the chemical changes that are associated with infection of human brain microvascular endothelial cells (hBMECs) by <i>T. gondii</i> (RH) tachyzoites. Both FTIR microspectroscopic methods showed utility in revealing the chemical alterations in the infected hBMECs. Using a ZnS hemisphere device, to increase the numerical aperture, and the synchrotron source to increase the brightness, we obtained spatially resolved spectra from within a single cell. The spectra extracted from the nucleus and cytosol containing the tachyzoites were clearly distinguished. RNA sequencing analysis of <i>T. gondii</i>-infected and uninfected hBMECs revealed significant changes in the expression of host cell genes and pathways in response to <i>T. gondii</i> infection. These FTIR spectroscopic and transcriptomic findings provide significant insight into the molecular changes that occur in hBMECs during <i>T. gondii</i> infection.