Ceramides (Cer) and dihydroceramides (dhCer) play critical roles in cell signaling, membrane stability and inflammation. Unlike Cer, dhCer is a relatively unexplored metabolite in sphingolipid (SL) metabolism, despite being an important intermediate of the de novo pathway in the endoplasmic reticulum (ER). Neisseria meningitidis (Nm), a human-specific pathogen that causes meningitis, can modulate SL metabolism in brain endothelial cells (BECs). We found that Nm infection significantly increased dhCer levels and sought to investigate the underlying cellular mechanisms. BECs (hCMEC/D3s) were infected with Nm, and activity of dhCer-desaturase 1 (DEGS1) was measured using deuterated dhCer with LC-MS/MS analysis. Data revealed reduced DEGS1 activity, explaining the observed dhCer accumulation. Transcriptional analysis of de novo enzymes showed no regulation, but downregulation of ceramidase ACER3, potentially contributing to dhCer increase, was detected. Protein levels of DEGS1 were unaltered. To investigate dhCer subcellular localization, we employed clickable azido-dihydrosphingosine in live-cell confocal microscopy. Plasma membrane (PM) staining was combined with transfection of mApple-Sec61b-C1 to detect ER localization. Immunofluorescence staining with GM-130 identified the Golgi apparatus, and biotin/streptavidin-AF 647 staining localized Nm. Interestingly, we observed that clicked SL species localized to the ER, followed by translocation to the PM via the Golgi apparatus. LC-MS/MS analysis of purified fractions of the PM, Golgi apparatus, vesicles and the supernatant confirmed the subcellular distribution of dhCer. Collectively, our results indicate that dhCer increase in BECs after Nm infection is mainly due to post-translational inhibition of DEGS1. We demonstrate that dhCer is transported from the ER to the PM via the Golgi apparatus in vesicles and subsequently released to the supernatant, suggesting potential interaction with the surrounding environment.