Spinocerebellar ataxia type 3 (SCA3) is the most common dominantly inherited ataxia, and biomarkers are needed to noninvasively monitor disease progression and treatment response. Anti-ATXN3 antisense oligonucleotide (ASO) treatment has been shown to mitigate neuropathology and rescue motor phenotypes in SCA3 mice. Here, we investigated whether repeated ASO administration reverses brainstem and cerebellar neurochemical abnormalities by magnetic resonance spectroscopy (MRS). Symptomatic SCA3 mice received intracerebroventricular treatment of ASO or vehicle and were compared to wild-type vehicle-treated littermates. To quantify neurochemical changes in treated mice, longitudinal 9.4T MRS of cerebellum and brainstem was performed. Acquired magnetic resonance (MR) group means were analyzed by 2-way analysis of variance mixed-effects sex-adjusted analysis with post hoc Sidak correlation for multiple comparisons. Pearson correlations were used to relate SCA3 pathology and behavior. MR spectra yielded 15 to 16 neurochemical concentrations in the cerebellum and brainstem. ASO treatment in SCA3 mice resulted in significant total choline rescue and partial reversals of taurine, glutamine, and total N-acetylaspartate across both regions. Some ASO-rescued neurochemicals correlated with reduction in diseased protein and nuclear ATXN3 accumulation. ASO-corrected motor activity correlated with total choline and total N-acetylaspartate levels early in disease. SCA3 mouse cerebellar and brainstem neurochemical trends parallel those in patients with SCA3. Decreased total choline may reflect oligodendrocyte abnormalities, decreased total N-acetylaspartate highlights neuronal health disturbances, and high glutamine may indicate gliosis. ASO treatment fully or partially reversed select neurochemical abnormalities in SCA3 mice, indicating the potential for these measures to serve as noninvasive treatment biomarkers in future SCA3 gene silencing trials. ANN NEUROL 2023;94:658-671.