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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 (<i>Perilla frutescens</i> 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.