ABSTRACT Introduction Illicit fentanyl exposure during pregnancy is associated with adverse outcomes. Yet its direct effects and their underlying molecular mechanisms in human trophoblasts remain largely unknown. Xylazine and medetomidine, two ɑ2-adrenergic agonists, were recently detected as adulterants in illicit fentanyl-containing products, and have been linked to worsened clinical outcomes. Methods We assessed the transcriptional responses of human trophoblast stem (TS) cells in their cytotrophoblast state (TS-CTBs) and derived syncytiotrophoblasts (STBs) after 4-day and 9-day exposure to fentanyl alone or combined with medetomidine and xylazine (FMX). Results Fentanyl alone produced modest gene expression changes in TS-CTBs, but consistently induced suppression of mitochondrial and ribosomal transcriptional programs in STBs regardless of exposure duration, an effect absent in parental TS-CTBs. 9-day FMX co-exposure influenced mitochondrial respiratory chain gene expression in derived STBs, resulting in discordant regulation of nuclear versus mitochondrial genome-encoded respiratory chain genes. Temporal pathway analysis reveals progressive trophoblast remodeling and dysregulation of mitochondrial transcriptional programs following fentanyl and FMX co-exposure. Discussion Together, these findings illuminate trophoblast pathways affected by fentanyl alone and by FMX co-exposure, and establish a basis for future studies examining the functional and clinical relevance of these transcriptional responses. Highlights Syncytiotrophoblasts are transcriptionally sensitive to fentanyl exposure Mitochondrial transcriptional programs are suppressed in drug-exposed trophoblasts FMX co-exposure reshapes fentanyl-associated transcriptional changes in trophoblasts