Real-time images of nitrogen fixation in an intact nodule of hydroponically cultured soybean (Glycine max [L] Merr.) were obtained. In the present study, we developed a rapid method to produce and purify 13N-labeled radioactive nitrogen gas (half life: 9.97 min). 13N was produced from a 16O (p, α) 13N nuclear reaction. The target chamber was filled with CO2 and irradiated for 10 min with protons at an energy of 18.3 MeV and an electric current of 5 μA, which was delivered from a cyclotron. All CO2 in the collected gas was absorbed and removed with powdered soda-lime in a syringe and replaced with helium gas. The resulting gas was injected into gas chromatography and separated and a 35 mL fraction, including the peak of [13N]-nitrogen gas, was collected by monitoring the chromatogram. The obtained gas was mixed with 10 mL of O2 and 5 mL of N2 and used in the tracer experiment. The tracer gas was fed into the underground part of intact nodulated soybean plants and serial images of the distribution of 13N were obtained non-invasively using a positron-emitting tracer imaging system (PETIS). The rates of nitrogen fixation of the six test plants were estimated to be 0.17 ± 0.10 μmol N2 h−1 from the PETIS image data. The decreasing rates of assimilated nitrogen were also estimated to be 0.012 ± 0.011 μmol N2 h−1. In conclusion, we successfully observed nitrogen fixation in soybean plants with nodules non-invasively and quantitatively using [13N]N2 and PETIS.