Lunasin, a bioactive peptide with a variety of physiological functions, was overexpressed in soybean to generate a transgenic soybean. Polymerase chain reaction (PCR) analysis suggested that lunasin was successfully inserted into the soybean genome, and three transgenic lines, L12, L43, and L45, were selected for further study. Lunasin expression was characterized in the lines by Western blot and ultra-performance liquid chromatography with tandem mass spectrometry. Enzyme-linked immunosorbent assay showed that lunasin content in L12, L43, and L45 lines was 1.47 mg g^-1, 1.32 mg g^-1 and 1.98 mg g^-1, respectively; these values were significantly higher than that in wild-type soybean (0.94 mg g^-1). Lunasin enrichments from transgenic soybean (LET) exhibited stronger DPPH, ABTS⁺, and oxygen radical scavenging activity than lunasin enrichments from wild-type soybean (LEW). Further, LET presented superior anti-inflammatory activity on lipopolysaccharide-induced macrophage cells compared to LEW, and it significantly suppressed the release of nitric oxide (NO) and pro-inflammatory cytokines including interleukin-1 and -6. Moreover, LET showed higher anti-proliferation activity on MDA-MB-231 cells than LEW. Immunofluorescence staining showed that LET could internalize into NIH-3T3 cells, and localize in the nucleus. In conclusion, it is feasible and efficient to produce lunasin through a transgenic soybean expression system. Lunasin overexpressing soybean could be consumed as a functional food in the diets of patients with cancer and obesity in the future.