Air temperature and light conditions are important factors not only to produce high-quality seedlings but also to promote energy efficiency in a plant factory with artificial lighting. In this study, we conducted two experiments in order to investigate the favorable conditions of air temperature, light intensity and photoperiod for the production of cucumber scions and rootstocks in a plant factory with artificial lighting. Cucumber scions and rootstocks were cultivated in two combined treatments: the combination of three different levels of difference between the day and night temperature (DIF), 25/20, 26/18 and 27/16 °C and five different light intensity conditions of photosynthetic photon flux, 50, 100, 150, 200 and 250 μmol·m−2·s−1 was set for the first experiment, and the combination of three different photoperiod conditions, 12, 16 and 20 h·d−1 and five different light intensity conditions, 50, 100, 150, 200 and 250 μmol·m−2·s−1 was set for the second experiment. In the air temperature and light intensity treatments, the hypocotyl elongation of cucumber scions and rootstocks was affected more largely by light intensity than DIF. The highest DIF treatment (27/16 °C) affected negatively on the accumulation of dry mass. On the contrary, the smallest DIF treatment (25/20 °C) was favorable for seedling growth due to lesser stress by rapid change of air temperature between photo- and dark-period. In the photoperiod and light intensity treatments, an increased DLI (daily light integral) promoted the growth of scions and rootstocks. Under the same DLI condition, the growth of scions and rootstocks increased with increasing photoperiod and decreasing light intensity. In both of experiments, while the dry weight increased with increasing the light intensity, the light use efficiencies were reduced by increasing the light intensity. Considering the growth and quality of seedlings and energy efficiency, the optimal environment conditions were represented by 25/20 °C of air temperature, 150 μmol·m−2·s−1 of light intensity and 16 h·d−1 of photoperiod.