Root temperature has long been considered an essential environmental factor influencing the plant’s physiology. However, little is known about the effect of root temperature on the quality of the food produced by the plant, especially that of horticultural crops. To fill this gap, two independent root cooling experiments (15 °C vs. 20 °C and 10 °C vs. 20 °C) were conducted in autumn 2017 and spring 2018 in hydroponics with Chinese broccoli (Brassica oleracea var. alboglabra Bailey) under greenhouse conditions. The aim was to investigate the effect of root temperature on plant growth (biomass, height, yield) and food quality (soluble sugars, total chlorophyll, starch, minerals, glucosinolates). A negative impact on shoot growth parameters (yield, shoot biomass) was detected by lowering the root temperature to 10 °C. Chinese broccoli showed no response to 15 °C root temperature, except for an increase in root biomass. Low root temperature was in general associated with a higher concentration of soluble sugars and total chlorophyll, but lower mineral levels in stems and leaves. Ten individual glucosinolates were identified in the stems and leaves, including six aliphatic and four indolic glucosinolates. Increased levels of neoglucobrassicin in leaves tracked root cooling more closely in both experiments. Reduction of root temperature by cooling could be a potential method to improve certain quality characters of Chinese broccoli, including sugar and glucosinolate levels, although at the expense of shoot biomass.