The growth of plants and their glucosinolate content largely depend on the cultivation environment; however, there are limited reports on the optimization of ambient environmental factors for kale grown in plant factories. This study was conducted to investigate the effects of temperature, relative humidity, and the carbon dioxide (CO2) concentration on kale growth and glucosinolate content in different growth stages of cultivation in a plant factory. Kale was grown under different temperatures (14, 17, 20, 23, and 26 °C), relative humidities (45, 55, 65, 75, and 85%), and CO2 concentrations (400, 700, 1000, 1300, and 1600 ppm) in a plant factory. Two and four weeks after transplantation, leaf samples were collected to evaluate the physical growth and glucosinolate contents. The statistical significance of the treatment effects was determined by two-way analysis of variance, and Duncan’s multiple range test was used to compare the means. A correlation matrix was constructed to show possible linear trends among the dependent variables. The observed optimal temperature, relative humidity, and CO2 range for growth (20–23 °C, 85%, and 700–1000 ppm) and total glucosinolate content (14–17 °C, 55–75%, and 1300–1600 ppm) were different. Furthermore, the glucosinolate content in kale decreased with the increase of temperature and relative humidity levels, and increased with the increase of CO2 concentration. Most of the physical growth variables showed strong positive correlations with each other but negative correlations with glucosinolate components. The findings of this study could be used by growers to maintain optimum environmental conditions for the better growth and production of glucosinolate-rich kale leaves in protected cultivation facilities.