The most significant breakthrough in plant biotechnology is the development of the techniques to transform genes from unrelated sources into commercially important crop plants to develop resistance against targeted insect pests. The spatio-temporal expression of insecticidal genes in transgenic cotton varies with plant age, plant parts and environmental conditions. The understanding of this temporal and spatial variation in efficacy and the resulting mechanisms is essential for cotton protection and production. This review summarizes variations in the efficacy of introduced insecticidal genes in cotton crop. The factors contributing to the variability of endotoxins have also been highlighted. The reduction in Bt protein biosynthesis in late-season cotton tissues could be attributed to the overexpression of the Bt gene at earlier stages, which leads to gene regulation at post-transcription levels and consequently results in gene silencing at a later stage. Methylation of the promoter may also play a role in the declined expression of endotoxin proteins. In genetically modified crops several environmental factors have been reported to affect the expression of transgenes. Among environmental factors nitrogen metabolism, inhibition of synthesis, degradation, remobilization and high temperature are attributable to the quantitative reduction in Bt proteins. Applying plant growth regulators or protein enhancers such as ChaperoneTM may improve Bt cotton efficacy through enhancing the synthesis of proteins. Also some agronomic practices such as nitrogen fertilization and timely irrigation favour the endotoxin expression. Thus, variations in the efficacy of insecticidal genes in transgenic cotton and the involved mechanisms need to be understood fully so as to plan rational resistance management strategies to retard the rate of resistance development and to control target pests effectively by enhancing the endotoxin expression through genetic or agronomic management.