Advanced prostate cancers (PCa) often metastasize to the bone, where they eventually develop resistance to virtually all available therapies. We previously reported that TGF-beta induces acetylation of the transcription factor KLF5 at K369, and acetylated KLF5 (Ac-KLF5) is essential for TGF-beta to maintain EMT, promote osteoclast differentiation, and cause bone metastasis in prostate cancer. On the other hand, tetracycline derivatives doxycycline (D) and methacycline (M) can inhibit tumor growth and even metastasis, but their molecular targets remain undetermined. In this study, we screened 1987 FDA-approved drugs to identify those that selectively bind to the KLF5 K369Q mutant, which mimics Ac-KLF5, using surface plasmon resonance assays. We identified D and M, but not their parent compound, tetracycline (T), as potential inhibitors of Ac-KLF5. Functionally, D and M but not T inhibited KLF5 K369Q -induced cell invasion, osteoclast differentiation, and osteolytic bone metastasis. RNA-seq and ChIP-seq analyses identified 22 genes transcriptionally regulated by KLF5K369Q via direct promoter binding, with transcription modulated by D and M but not T. One gene, PGK1, was shown to be responsible for D- and M-suppressed cell invasion, osteolytic differentiation, and bone metastasis. These findings suggest a drug-target relationship between doxycycline/methacycline and Ac-KLF5, providing a therapeutic strategy for treating prostate cancer bone metastases resistant to antiandrogen therapy and chemotherapy.