The aim of this study was to investigate the bioavailability and bioactivity of perilla (Perilla frutescens) oil nanoemulsions prepared at different homogenization pressures by measuring the weight, fatty acid profile, and antioxidant and anti-inflammatory properties in rats. The high-pressure homogenization significantly reduced the particle size of perilla oil nanoemulsions and enhanced their stability, and the minimum particle size was 293.87 ± 6.55 nm at 120 MPa. There was an increase in the weight and fatty acid levels in the plasma and liver of test group rats. The highest glutathione (GSH) and the lowest malondialdehyde (MDA) levels of 18.76 ± 10.51 mg GSH/g prot and 20.27 ± 2.46 nmol/mg prot, respectively, were recorded in rats administrated perilla oil nanoemulsions prepared at 120 MPa. However, there was no significant difference in superoxide dismutase activity (SOD) between the groups. The interferon-gamma (IL-γ), interleukin-1 beta (IL-1β), IL-6 (interleukin-6), and IL-8 (interleukin-8) levels in the test groups were lower than those in the blank and control groups at 8 hr after lipopolysaccharide injection. The IL-1β, IL-6, and IL-8 levels were 49.52 ± 14.06, 90.13 ± 6.04, and 419.71 ± 32.03 ng/L, respectively, in rats treated with perilla oil nanoemulsions prepared at 120 MPa. Both perilla oil and its nanoemulsions decreased estradiol levels and damaged the ovaries. Overall, our findings show that the test nanoemulsions enhanced the bioavailability of perilla oil, which resulted in enhanced antioxidant and anti-inflammatory responses; thus, we provide a new approach to deliver perilla oil. PRACTICAL APPLICATION: Nanoemulsions can be used to deliver drugs and bioactive compounds, and perilla oil nanoemulsions can be used in healthcare products and beverage industries.