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As an innovative technology in biological applications—non-thermal plasma technique—has recently been applied to living cells and tissues. However, it is unclear whether non-thermal plasma treatment can directly regulate the growth and development of livestock. In this study, we exposed four-day-incubated fertilized eggs to plasma at 11.7 kV for 2 min, which was found to be the optimal condition in respect of highest growth rate in chickens. Interestingly, plasma-treated male chickens conspicuously grew faster than females. Plasma treatment regulated the reactive oxygen species homeostasis by controlling the mitochondrial respiratory complex activity and up-regulating the antioxidant defense system. At the same time, growth metabolism was improved due to the increase of growth hormone and insulin-like growth factor 1 and their receptors expression, and the rise of thyroid hormones and adenosine triphosphate levels through the regulation of demethylation levels of growth and hormone biosynthesis-related genes in the skeletal muscles and thyroid glands. To our knowledge, this study was the first to evaluate the effects of a non-thermal plasma treatment on the growth rate of chickens. This safe strategy might be beneficial to the livestock industry. [ABSTRACT FROM AUTHOR]