BackgroundTrichomoniasis caused byTrichomonas vaginalis, combined with its complications, has long frequently damaged millions of human health. Metronidazole (MTZ) is the first choice for therapy. Therefore, a better understanding of its trichomonacidal process to ultimately reveal the global mechanism of action is indispensable. To take a step toward this goal, electron microscopy and RNA sequencing were performed to fully reveal the early changes inT. vaginalisat the cellular and transcriptome levels after treatment with MTZ in vitro.ResultsThe results showed that the morphology and subcellular structures ofT. vaginalisunderwent prominent alterations, characterized by a rough surface with bubbly protrusions, broken holes and deformed nuclei with decreased nuclear membranes, chromatin and organelles. The RNA-seq data revealed a total of 10,937 differentially expressed genes (DEGs), consisting of 4,978 upregulated and 5,959 downregulated genes. Most DEGs for the known MTZ activators, such as pyruvate:ferredoxin oxidoreductase (PFOR) and iron-sulfur binding domain, were significantly downregulated. However, genes for other possible alternative MTZ activators such as thioredoxin reductase, nitroreductase family proteins and flavodoxin-like fold family proteins, were dramatically stimulated. GO and KEGG analyses revealed that genes for basic vital activities, proteostasis, replication and repair were stimulated under MTZ stress, but those for DNA synthesis, more complicated life activities such as the cell cycle, motility, signaling and even virulence were significantly inhibited inT. vaginalis.Meanwhile, increased single nucleotide polymorphism (SNP) and insertions - deletions (indels) were stimulated by MTZ.ConclusionsThe current study reveals evident nuclear and cytomembrane damage and multiple variations inT. vaginalisat the transcriptional level. These data will offer a meaningful foundation for a deeper understanding of the MTZ trichomonacidal process and the transcriptional response ofT. vaginalisto MTZ-induced stress or even cell death.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09339-9.