In this study, we investigated the effect of UV-B irradiation 3 days prior to harvest, on the accumulation of rosmarinic acid (RA) and anthocyanin, and the expression of genes related to phenylpropanoid and flavonoid biosynthetic pathways, in red perilla (Perilla frutescens L.). In experiment 1, seedlings at 60 days after sowing (DAS) were subjected to UV-B irradiation at 0 (control), 6, and 10 W m−2 under a 16 h light period; while in experiment 2, seedlings at 45 DAS were subjected to UV-B irradiation at 0 (control), 4 W m−2 at continuous irradiation, and 6 W m−2 at 16 h irradiation. UV irradiation of 10 W m−2 for 16 h negatively affected leaf color, while irradiation at 6 W m−2 enhanced RA biosynthesis and antioxidant capacity. Continuous UV-B irradiation of 4 W m−2 increased the RA concentration by 92% compared to the control; however, this effect was smaller than that of UV-B irradiation at 6 W m−2 for 16 h, 141% higher than that of the control and had a lower antioxidant capacity against UV-mediated ROS overproduction during the dark period. Results demonstrate that 6 W m−2 of UV-B irradiation for 16 h is suitable for enhancing the RA concentration and content of red perilla.