The signal transducer and activator of transcription 3 (STAT3) are ideal targets for ovarian cancer. Previous studies showed that downregulation of STAT3 using specific short hairpin RNAs (shRNA) can significantly reduce ovarian tumor growth. However, RNA interference does not fully ablate target gene expression due to idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of STAT3-specific shRNA, we employed a combinatorial expression of STAT3-specific shRNA and liver kinase B1 (LKB1), a tumor suppressor. Thus, the LKB1 coding sequences and STAT3-specific shRNAs were constructed in a eukaryotic co-expression plasmid pCDNA3.1, and then transfected into ovarian cancer cells to evaluate the synergistic effects of this combination on anticancer activity and explore the relevant molecular mechanisms. Co-expression of STAT3‑specific siRNA and LKB1 (pSi-STAT3-LKB1) synergistically inhibited ovarian cancer cell growth, invasion and migration, induced cell apoptosis and arrested the cell cycle in vitro when compared with monotherapy. The results showed that the co-expression of plasmid pSi-STAT3-LKB1 inserted subcutaneously into ovarian tumor xenograft resulted in more significant inhibition of tumor growth. Further study showed that the synergistic anti-ovarian cancer effects of the co-expression of STAT3-specific siRNA and LKB1 may be associated with the upregulation of p-p53, p21 and downregulation of survivin, BCL-2 and cyclin D1. Results of the present study suggested that combined therapy with eukaryotic co-expression of the plasmid‑carrying STAT3-specific siRNA and LKB1 is a novel and efficient treatment strategy for human ovarian cancer.