The Weakly Interacting Massive Particles (WIMPs) are considered to be the hypothetical particles that have been the leading candidate for dark matter for decades. The PICOLON project is directly searching for WIMPs dark matter using ultra-pure NaI(Tl) crystals at the Kamioka underground laboratory. To determine the WIMP sensitivity, it is necessary to know the quenching factor (QF) of NaI(Tl), which is the scintillation light yield ratio of nuclear recoil and electron recoil at the same energy deposit. Several groups reported the QF absolute values and its energy dependence in the low energy region (less than 100 keV), however the results are not in good agreement. It is still unsolved whether the disagreement is due to the individual crystal differences or the effect of systematic errors. In this paper, we report the QF of ultra-pure NaI(Tl) crystal developed by PICOLON group. To measure the QF, the crystal was irradiated with 2.45-MeV monochromatic neutrons generated by a discharge-type compact D-D fusion neutron source at the Institute of Advanced Energy, Kyoto University. The QFs of Na were obtained at six points in the range of 19–101 keVnr.