Global climate change and the industrial revolution have increased the concentration of tropospheric ozone, a photochemical air pollutant that can negatively affect plant growth and crop production. In the present study, we investigated the effects of O3 on the metabolites and transcriptome of tartary buckwheat. A total of 37 metabolites were identified by gas chromatography coupled with time-of-flight mass spectrometry, and principal component analysis was performed to verify the metabolic differences between non-treated and O3-treated Tartary buckwheat. The levels of most metabolites (threonic acid, tryptophan, sucrose, and raffinose) decreased significantly after the O3 treatment. On the contrary, the levels of two anthocyanins, cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside, increased more than 11.36- and 11.43-fold, respectively after the O3 treatment. To assess the effect of O3 on the genomic level, we analyzed the expression of anthocyanin biosynthesis pathway genes in O3-treated and non-treated buckwheat using quantitative real-time reverse transcription PCR. We found that the expression of all anthocyanin pathway genes increased significantly in the O3-treated buckwheat compared to that in the non-treated buckwheat. Altogether, our results suggested that O3 affected the transcripts and metabolites of tartary buckwheat, which would eventually cause phenotypic changes in plants.