Transcription factors forkhead box (Fox)O1 and pancreatic and duodenal homeobox-1 (PDX-1) are involved in dexamethasone (DEX)-induced dysfunction in pancreatic β-cells. However, the molecular mechanism underlying the regulation of FoxO1 and PDX-1 expression in β-cells treated with DEX is not fully understood. In this study, we found that DEX markedly increased FoxO1 mRNA and protein expression, whereas it decreased PDX-1 mRNA and protein expression in a dose- and time-dependent manner. Further study showed that FoxA2 was involved in regulation of FoxO1 and PDX-1 expression in DEX-induced pancreatic β-cells dysfunction. Interestingly, we demonstrated for the first time that FoxA2 could bind to the FoxO1 gene promoter and positively regulate FoxO1 expression. Moreover, we found that DEX increased the activity of FoxA2 binding to the FoxO1 promoter but decreased the activity of FoxA2 binding to the PDX-1 promoter of RINm5F cells. Knockdown of FoxA2 by RNA interference inhibited FoxO1 expression and restored PDX-1 expression in pancreatic β-cells treated with DEX. However, DEX had no effect on the expression of FoxA2. Together, the results of the present study demonstrated that FoxA2 could dynamically regulate FoxO1 and PDX-1 expression in pancreatic β-cells treated with DEX, which provides new important information on the transcriptional regulation of FoxO1 and PDX-1 in DEX-induced pancreatic β-cells. Inhibition of FoxA2 can effectively protect β-cells against DEX-induced dysfunction.