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The aim of the present study was to evaluate the effects of alternating red (660 nm) and blue (460 nm) light on the growth and nutritional quality of two-leaf-color pak choi (Brassica campestris L. ssp. chinensis var. communis). Four light treatments (supplemental alternating red and blue light with intervals of 0, 1, 2, and 4 hours, with a monochromatic light intensity of 100 μmol·m−2·s−1 and a cumulative lighting time of 16 hours per day) were conducted in a greenhouse under identical ambient light conditions (90 to 120 μmol·m−2·s−1 at 12:00 am) for 10 days before green- and red-leaf pak choi were harvested. The results showed that the two-leaf-color pak choi receiving alternating red and blue light exhibited more compact canopies and wider leaves than those under the control treatment, which was attributed to the shade avoidance syndrome of plants. The present study indicated that the biomass of green-leaf pak choi was much higher than that of red-leaf pak choi, but the nutritional quality of green-leaf pak choi was lower than that of red-leaf pak choi, and seemingly indicating that the regulation of metabolism for pak choi was species specific under light exposure. The trends of both biomass and the soluble sugar content were highest under the 1-hour treatment. The contents of chlorophyll a and total chlorophyll in both cultivars (green- and red-leaf pak choi) were significantly increased compared with control, without significant differences among the 1-, 2-, and 4-hour treatments, whereas chlorophyll b exhibited no significant difference in any treatment. Alternating red- and blue-light treatment significantly affected the carotenoid content, but different trends in green- and red-leaf pak choi were observed, with the highest contents being detected under the 1-hour and 4-hour treatments, respectively. With increasing time intervals, the highest soluble protein contents in two-leaf-color pak choi were observed in the 4-hour treatment, whereas nitrate contents were significantly decreased in the 4-hour treatment. Compared with 0 hours, the contents of vitamin C, phenolic compounds, flavonoids, and anthocyanins in two-leaf-color pak choi were significantly increased, but no significant differences were observed in vitamin C, phenolic compounds, and flavonoids among the 1-, 2-, and 4-hour treatments, similar to what was found for the anthocyanin content of green-leaf pak choi. However, the content of anthocyanins in red-leaf pak choi gradually increased with increasing time intervals, with the highest content being found in the 4-hour treatment. Supplemental alternating red and blue light slightly increased the antioxidant capacity [1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rate and antioxidant power], but no significant differences were observed after 1, 2, and 4 hours of treatment. Taken together, treatment with an interval of 1 hour was the most effective for increasing the biomass of pak choi in this study, but treatment with a 4-hour interval should be considered to enhance the accumulation of health-promoting compounds.