The present study evaluated effects of β-sitosterol on growth performance, meat quality, oxidative status, and mitochondrial biogenesis of breast muscle in broilers. One-day-old chicks were allocated to five treatments of six replicates. Broilers were fed a basal diet supplemented either with 0 (control), 40, 60, 80, or 100 mg/kg β-sitosterol for 42 days. β-sitosterol linearly and quadratically reduced feed/gain ratio, lightness24h and cooking loss24h in breast muscle, whereas 2, 2-diphenyl-1-picrylhydrazyl scavenging activity of breast muscle followed an opposite trend. β-sitosterol linearly decreased drip loss24h and malondialdehyde content, whereas linearly increased pH24h, superoxide dismutase activity, and mRNA abundances of peroxisome proliferator-activated receptor γ coactivator 1α (PCG-1α) and mitochondrial transcription factor A (TFAM) in breast muscle. Compared with control, levels of β-sitosterol higher than 40 mg/kg reduced feed/gain ratio, muscular lightness24h, cooking loss24h, and malondialdehyde level, whereas increased muscular 2, 2-diphenyl-1-picrylhydrazyl scavenging activity, and mRNA abundances (except 60 mg/kg) of PCG-1α and TFAM. Eighty milligram/kilogram β-sitosterol increased muscular pH24h and superoxide dismutase activity, but decreased its drip loss24h. Therefore, β-sitosterol could improve growth performance and meat quality, oxidative status, and mitochondrial biogenesis of breast muscle in broilers. Furthermore, supplementation level of 80 mg/kg β-sitosterol is recommended for broiler diets.