Hemp (Cannabis sativa) has gained global attention since being legalized in the USA in 2018. The legalization of hemp is also underway in Korea; however, it requires facility cultivation and an agricultural technological system that can produce high-quality plants. This study investigated the changes in the growth and secondary metabolite content of female hemp following treatment with the plant growth retardant diniconazole (DIN). Treatment with DIN decreased the plant growth rate with increasing concentrations; however, at a concentration of 25 mg∙L⁻¹, it increased inflorescence biomass and apical inflorescence size. The high-performance liquid chromatograph analysis of major cannabinoids showed no statistically significant differences in total cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC) contents among the treatment groups (25, 50, 100, 200, and 400 mg∙L⁻¹). However, the calculated production of CBD and THC per plant was significantly highest at DIN 25 mg∙L⁻¹. Overall, at a concentration of 25 mg∙L⁻¹, DIN treatment not only decreased the height of female hemp but also enhanced the production of female hemp inflorescences as well as major cannabinoids (CBD and Δ9-THC). Our results indicate that at a concentration of 25 mg∙L⁻¹, DIN is suitable for use in a vertical farming system. However, the Δ9-THC content needs to meet the 0.3% standard for industrial use. These findings can be applied in various fields that use hemp.