In this work, the effects of the protozoan Neospora caninum on the bioenergetics, chemical composition, and elemental content of human brain microvascular endothelial cells (hBMECs) were investigated. We showed that N. caninum can impair cell mitochondrial (Mt) function and causes an arrest in host cell cycling at S and G2 phases. These adverse effects were also associated with altered expression of genes involved in Mt energy metabolism, suggesting Mt dysfunction caused by N. caninum infection. Fourier Transform Infrared (FTIR) spectroscopy analysis of hBMECs revealed alterations in the FTIR bands as a function of infection, where infected cells showed alterations in the absorption bands of lipid (2924 cm−1), amide I protein (1649 cm−1), amide II protein (1537 cm−1), nucleic acids and carbohydrates (1092 cm−1, 1047 cm−1, and 939 cm−1). By using quantitative synchrotron radiation X-ray fluorescence (μSR-XRF) imaging and quantification of the trace elements Zn, Cu and Fe, we detected an increase in the levels of Zn and Cu from 3 to 24 h post infection (hpi) in infected cells compared to control cells, but there were no changes in the level of Fe. We also used Affymetrix array technology to investigate the global alteration in gene expression of hBMECs and rat brain microvascular endothelial cells (rBMVECs) in response to N. caninum infection at 24 hpi. The result of transcriptome profiling identified differentially expressed genes involved mainly in immune response, lipid metabolism and apoptosis. These data further our understanding of the molecular events that shape the interaction between N. caninum and blood-brain-barrier endothelial cells.