The highly conserved RNA-binding protein LIN28B and focal adhesion kinase (FAK) are significantly upregulated in ovarian cancer (OC), serving as markers for disease progression and prognosis. Nonetheless, the correlation between LIN28B and FAK, as well as the pharmacological effects of the LIN28 inhibitor C1632, in OC cells have not been elucidated. The present study demonstrates that C1632 significantly reduced the rate of DNA replication, arrested the cell cycle at the G0/G1 phase, consequently reducing cell viability, and impeding clone formation. Moreover, treatment with C1632 decreased cell-matrix adhesion, as well as inhibited cell migration and invasion. Further mechanistic studies revealed that C1632 inhibited the OC cell proliferation and migration by concurrently inhibiting LIN28 B/let-7/FAK signaling pathway and FAK phosphorylation. Furthermore, C1632 exhibited an obvious inhibitory effect on OC cell xenograft tumors in mice. Altogether, these findings identified that LIN28 B/let-7/FAK is a valuable target in OC and C1632 is a promising onco-therapeutic agent for OC treatment.