Cervical cancer is one of the most prevalent malignancies in women worldwide. Therefore, investigation about molecular pathogenesis and related therapy targets of cervical cancer is an emergency. The molecular mechanisms responsible for the chemoresistance of cervical cancer were investigated by the use of doxorubicin (Dox)-resistant HeLa/Dox and SiHa/Dox cells. Our data showed that chemoresistant cells exhibited significantly higher glucose consumption, lactate production rate, and ATP levels than that of their parental cells. Among metabolic and glycolytic related genes, the expression of PDK4 was upregulated in Dox-resistant cells. Knockdown of PDK4 can decrease glucose consumption, lactate production rate, and ATP levels and further sensitize resistant cervical cancer cells to Dox treatment. By screening microRNAs (miRNAs), which can regulate expression of PDK4, we found that miR-16-5p was downregulated in chemoresistant cells. Overexpression of miR-16-5p can decrease the expression of PDK4 and sensitize the resistant cells to Dox treatment. Xenograft models confirmed that knockdown of PDK4 can increase chemotherapy efficiency for in vivo tumor growth. Collectively, our data suggested that miR-16-5p/PDK4-mediated metabolic reprogramming is involved in chemoresistance of cervical cancer.