[Objective] The study aimed to determine the appropriate stage for exploring the response of Bacillus thuringiensis to the alkaline stress, to profile the metabolic pathways under this stress. [Methods] Using semiquantitative RT-PCR and qRT-PCR, the proper stage was defined by monitoring the transcriptional changes of marker gene pspA, which was known as a responsive gene under the alkaline stress. The total RNA was then extracted to perform the microarray hybridizations for samples under stress and control, respectively. Gene Ontology and pathway enrichments were conducted to analyze the global changes of carbon metabolism, metabolism of fatty acid synthesis and amino acid. [Results] For B. thuringiensis in the mid-log growth phase, treatment of 28 mmol/L NaOH for 10 mins is the feasible approach to analyze the response of B. thuringiensis to this stress. More than twenty genes encoding important enzymes in glycolytic pathway were up-regulated and majority of genes involved in catalyzing alpha-ketoglutarate into malic acid were also found to up-regulated more than two folds. [Conclusion] By analyzing the gene expression profile, the major metabolisms of B. thuringiensis were found to be clearly enhanced under alkaline stress. Large quantities of acid including malic acid and lactic acid may contribute a lot to the adaptation of alkaline condition.