The impact of 9 months cultivation of transgenic Bacillus thuringiensis (Bt) cotton on selected culturable bacterial and fungal populations in rhizosphere soil was investigated. The transgenic Bt cotton line (06Z604M), isoline (99M03) and a non-Bt cotton line (HART 89M) were planted in confined field site in Samuru-Thika where non-Bt cotton had been continuously cultivated for the past 2 years. Rhizosphere soil samples were collected at planting, maturity, flower and boll, and boll-opening stages and harvesting stages of cotton. Numbers of culturable soil microbial groups (bacteria, actinomycetes and fungi) involved in decomposition and nutrients recycling were measured at CFU and population levels. The proteins did not show effects on bacterial, actinomycetes and fungal counts and populations possibly as a result of adsorption of the proteins on soil particles, which could have rendered the proteins inaccessible for microbial utilization. Culturable microbial population and colony counts arranged in decreasing order were 06Z604D>99M03>HART89M, similar to the amounts of MBC and clay in the soils. Moreover, bacteria and fungi counts were higher at 110 DAS in 06Z604D than in 99M03 and HART89M plot soils. Our observations suggest that insecticidal proteins (Cry1Ac and Cry2Ab2) produced by Bollgard II Bt cotton could persist in tropical soils as a result of adsorption on soil clays but that there were no observable effect on the studied culturable microbial groups.The data presented here showed no consistent statistically significant differences (p<0.005) in the numbers of different groups of culturable bacteria, actinomycetes and fungi between rhizosphere soil of Bt, isoline and Hart 89M cotton lines in the same field, and no obvious trends in the numbers of the culturable bacteria and fungi with the increasing growth duration. Moreover, the studied culturable bacterial and fungal groups were positively correlated (p>0.001) with soil respiration and microbial biomass, which exhibited uneven trend with the treatments. Generally Soil from 06Z604D showed the slight higher microbial populations and CFU count, whilst HART 89M showed slight lower microbial count. This depicts the fact that slight variability in the treatments, quality and content of the root exudates might have a temporal or permanent shift in micro biota populations of a variety of crop studied.This study therefore suggests that a single-year cultivation of transgenic Bt cotton may not affect the functional bacterial and fungi populations in rhizosphere soil.