Mobility data are crucial for understanding the dynamics of coronavirus disease 2019 (COVID-19), but the consistency of the usefulness of these data over time has been questioned. The present study aimed to reveal the relationship between the transmissibility of COVID-19 in Tokyo, Osaka, and Aichi prefectures and the daily night-time population in metropolitan areas belonging to each prefecture. In Japan, the de facto population estimated from GPS-based location data from mobile phone users is regularly monitored by Ministry of Health, Labor, and Welfare and other health departments. Combined with this data, we conducted a time series linear regression analysis to explore the relationship between daily reported case counts of COVID-19 in Tokyo, Osaka, and Aichi, and night-time de facto population in downtown areas estimated from mobile phone location data, from February 2020 to May 2022. As an approximation of the effective reproduction number, the weekly ratio of cases was used. Models using night-time population with lags ranging from 7 to 14 days were tested. In time-varying regression analysis, the night-time population level and the daily change in night-time population level were included as explanatory variables. In the fixed-effect regression analysis, the inclusion of either the night-time population level or daily change, or both, as explanatory variables was tested, and autocorrelation was adjusted by introducing first-order autoregressive error of residuals. In both regression analyses, the lag of night-time population used in best fit models was determined using the information criterion. In the time-varying regression analysis, night-time population level tended to show positive to neutral effects on COVID-19 transmission, whereas the daily change of night-time population showed neutral to negative effects. The fixed-effect regression analysis revealed that for Tokyo and Osaka, regression models with 8-day-lagged night-time population level and daily change were the best fit, whereas in Aichi, the model using only the 9-day-lagged night-time population level was the best fit using the widely applicable information criterion. For all regions, the best-fit model suggested a positive relationship between night-time population and transmissibility, which was maintained over time. Our results revealed that, regardless of the period of interest, a positive relationship between night-time population levels and COVID-19 dynamics was observed. The introduction of vaccinations and major outbreaks of Omicron BA. Two subvariants in Japan did not dramatically change the relationship between night-time population and COVID-19 dynamics in three megacities in Japan. Monitoring the night-time population continues to be crucial for understanding and forecasting the short-term future of COVID-19 incidence.