Roehybridine β-N-oxide (RNO) is a proaporphine-tryptamine dimer alkaloid froti Roemeria hybrida. RNO possesses five stefeogenic centers and so far. its absolute configuration has not been determined. In this study, the absolute configuration of RNO was established as 6aS,7aR,6R,9S,1OR by comparing experimental and TDDFT calculated electronic circular dichroism (ECD) spectra. In addition, we have reported an amendment indicating exact (1)H and (13)C chemical shifts of RNO. RNO exhibited cytotoxic activity when tested in vitro against prostate cancer cell lines including PC3 and DU145. The IC(50) values of RNO against PC3 and DU145 were 13.3 and 15.1 μg/mL, respectively.