We evaluated the effect of transgenic cottons (Bacillus thuringiensis (Bt) and Bt with cowpea trypsin inhibitor (Bt–CpTI)) on soil ecosystems after three and four years of growth. The persistence and the relationships among insecticidal crystal proteins 1Ac (Cry1Ac) and cowpea trypsin inhibitor (CpTI) proteins derived from transgenic cottons, microbial properties, and enzymatic activities in the rhizosphere soil were investigated in the repeated cultivation experiment. The Cry1Ac and CpTI proteins persisted in the rhizosphere soil of transgenic cotton, whereas no proteins were detected in the rhizosphere soil of non-transgenic cotton. The persistence of CpTI inhibited the degradation of Cry1Ac protein in the rhizosphere soil of Bt–CpTI cotton. Repeated measures analysis of variance indicated that the year of cultivation, the variety of cotton, the transgenic trait, and their interactions significantly influenced the microbial properties and the enzymatic activities in rhizosphere soil. Overall, the repeated cultivation of transgenic cotton had significantly negative effects on the microbial properties and the enzymatic activities in rhizosphere soil compared to those in the rhizosphere soil of non-transgenic cotton.