Trichomonas vaginalisis the most common non-viral sexually transmitted infection. 5-nitroimidazoles are the only FDA-approved medications forT. vaginalistreatment. However, 5-nitroimidazole resistance has been increasingly recognized and may occur in up to 10% of infections. We aimed to delineate mechanisms ofT. vaginalisresistance using transcriptome profiling of metronidazole (MTZ)-resistant and sensitiveT. vaginalisclinical isolates. In vitro, 5-nitroimidazole susceptibility testing was performed to determine minimum lethal concentrations (MLCs) forT. vaginalisisolates obtained from women who had failed treatment (n= 4) or were successfully cured (n= 4). RNA sequencing, bioinformatics, and biostatistical analyses were performed to identify differentially expressed genes (DEGs) in the MTZ-resistant vs. sensitiveT. vaginalisisolates. RNA sequencing identified 304 DEGs, 134 upregulated genes and 170 downregulated genes in the resistant isolates. Future studies with moreT. vaginalisisolates with a broad range of MLCs are needed to determine which genes may represent the best alternative targets in drug-resistant strains.