The production cross-section of the medical isotope, ⁹⁹Mo from the enriched ¹⁰⁰Mo(n,2n) reaction with the average neutron energies of 21.9 and 26.5 MeV has been determined for the first time by using an off-line γ-ray spectrometric technique. The average neutron energies were generated by using the ⁹Be(p,n) reaction with the proton energies of 35 and 45 MeV from the MC50 cyclotron of the Korea Institute of Radiological and Medical Sciences (KIRAMS) at Seoul, South Korea. The ¹⁰⁰Mo(n,2n) reaction cross-section as a function of neutron energy was also calculated theoretically by using the computer code TALYS-1.8 and EMPIRE-3.2 Malta. The experimental results are in close agreement with the theoretical values from TALYS-1.8. However, the present data at the neutron energy of 21.9 MeV is slightly lower and at 26.5 MeV is higher than the values from EMPIRE-3.2 Malta.