As an indispensable element in the morphology and phytochemical profile of plants, UV-A has proved to help promote the growth and quality of kale. In this study, UV-A supplementation in different photoperiods (light period supplemental UVA = LS, dark period supplemental UVA = DS, and light-dark period supplemental UVA = LDS) contributed to yielding greater biomass production (fresh weight, dry weight, and plant moisture content), thus improving morphology (plant height, stem diameter, etc.) and promoting higher phytochemicals content (flavonoids, vitamin c, etc.), especially glucosinolates. To fathom its mechanisms, this study, using RNA-seq, verified that UV-A supplementation treatments signally generated related DEGs of plant hormone signal pathway, circadian rhythm plant pathway, glucosinolate pathway, etc. Moreover, 2047 DEGs were obtained in WGCNA, illustrating the correlations between genes, treatments, and pathways. Additionally, DS remarkedly up-regulated related DEGs of the key pathways and ultimately contributed to promoting the stem diameter, plant height, etc., thus increasing the pigment, biomass, vitamin c, etc., enhancing the antioxidant capacity, and most importantly, boosting the accumulations of glucosinolates in kale. In short, this study displayed new insights into UV-A supplementation affected the pathways related to the morphology and phytochemical profile of kale in plant factories.