Bt transgenic cotton has shown unstable insect-resistance during boll period in cotton industry. The objective of this study was to investigate the effect of the relative humidity on the leaf Bt protein content of two different types of Bt cotton cultivars under high temperature condition. During the 2009 and 2010 cotton growth seasons, the leaves insecticidal protein concentrations of the two Bt cultivars were measured under high (water-sprayed every day) and low field relative humidity (no water spray) during the high temperature period in three field experimental sites. In 2010, the environmentally controlled experiment with 3 humidity (50%, 70% and 90%) treatments under 37 °C was conducted to further investigate the effects of the relative humidity on the Bt efficacy and mechanism of nitrogen metabolism at Yangzhou University Farm. The results of the study showed that the reduction of cotton field RH under high temperature further decreased the leaf Bt insecticidal protein levels. The high RH treatment at boll developing period caused relatively less and slower decline of the insecticidal protein. In contrast, the low RH treatment had greater and faster drop of the Bt efficacy. The green house experiment further indicated that the greater reduction of the leaf entoxin was detected under extreme humidity(RH 90% and RH 50%) under high temperature compared to RH 70%. Correspondingly, the higher leaf amino acid contents, lower soluble protein levels and the greater protease activities under the extreme humidity than the RH 70% treatment were observed during the whole treatment period. It indicated that the reduced insect-resistance of Bt cotton that is closely correlated to extreme weather during boll developing period caused by the interaction of high temperature and extreme relative humidity. Leaf soluble protein reduction caused by extreme relative humidity under high temperature may contribute to a further decrease of the Bt toxin in the boll period of Bt cotton.