Earth's middle and upper atmosphere exhibits several dominant large-scale oscillations in many measured parameters. One of these oscillations is the semi-annual oscillation (SAO). The SAO can be detected in the ionospheric total electron content (TEC), the ionospheric transition height, the wind regime in the mesosphere-lower thermosphere (MLT), and in the MLT temperatures. In addition, as we report for the first time in this study, the SAO is among the most dominant oscillations in nighttime very low frequency (VLF) narrowband (NB) subionospheric measurements. As VLF signals are reflected off the ionospheric D region (at altitudes of ∼65 and ∼85 km, during the day and night, respectively), this implies that the upper part of the D region is experiencing this oscillation as well, through changes in the dominating electron or ion densities, or by changes in the electron collision frequency, recombination rates, and attachment rates, all of which could be driven by oscillatory MLT temperature changes. We conclude that the main source of the SAO in the nighttime D region is NOx molecule transport from the lower levels of the thermosphere, resulting in enhanced ionization and the creation of free electrons in the nighttime D region, thus modulating the SAO signature in VLF NB measurements. While the cause for the observed SAO is still a subject of debate, this oscillation should be taken into account when modeling the D region in general and VLF wave propagation in particular.