OBJECTIVES: The aim of this study was to explore whether indoor or outdoor relative humidity (RH) modulates the influenza epidemic transmission in temperate and subtropical climates. METHODS: In this study, the daily temperature and RH in 1558 households from March 2017 to January 2019 in five cities across both temperate and subtropical regions in China were collected. City-level outdoor temperature and RH from 2013 to 2019 were collected from the weather stations. We first estimated the effective reproduction number (R(t)) of influenza and then used time-series analyses to explore the relationship between indoor/outdoor RH/absolute humidity and the R(t) of influenza. Furthermore, we expanded the measured 1-year indoor temperature and the RH data into 5 years and used the same method to examine the relationship between indoor/outdoor RH and the R(t) of influenza. RESULTS: Indoor RH displayed a seasonal pattern, with highs during the summer months and lows during the winter months, whereas outdoor RH fluctuated with no consistent pattern in subtropical regions. The R(t) of influenza followed a U-shaped relationship with indoor RH in both temperate and subtropical regions, whereas a U-shaped relationship was not observed between outdoor RH and R(t). In addition, indoor RH may be a better indicator for R(t) of influenza than indoor absolute humidity. CONCLUSION: The findings indicated that indoor RH may be the driver of influenza seasonality in both temperate and subtropical locations in China.