Per- and polyfluoroalkyl substances (PFAS) are persistent pollutants known for their bio-accumulative properties, prevalence in household products, and widespread contamination of drinking water supplies. Although legacy PFAS, such as perfluorooctanoic acid, have been phased out in the U.S. due to public health concerns, a PFAS variant hexafluoropropylene oxide-dimer acid (HFPO-DA) is an emerging replacement. HFPO-DA is a potential neurotoxicant that has been shown to cause dopaminergic neurodegeneration. In this study, we investigated the bio-accumulative potential of HFPO-DA and its sex-specific effects on lifespan, locomotion, and brain gene expression in fruit flies. We utilized established lethality and locomotor assays to determine dose-response for HFPO-DA. We utilized a novel high-throughput, low-depth RNA sequencing method (shallow RNA- sequencing) that enables transcriptional profiling at a low cost per sample, to screen for exposure-affected genes in fly brains. We found that while HFPO-DA does not readily bioaccumulate in fruit fly bodies, high-dose exposures have sex-specific effects on lifespan, locomotor ability, and brain gene expression. The LOAEL (lowest adverse effect level) for median lifespan was 10 mg/kg/day in females, and 102 mg/kg/day in males. Both locomotor ability and brain gene expression exhibited non-conventional dose-response, similar to patterns reported endocrine disrupting chemical exposures.