Background: Prostate cancer is a life-threating disease with high incidence and mortality in male. Formononetin, the main active component of some natural products, has been hypothesized as a promising anticancer agent in previous studies. Objectives: We investigated the toxic effects and potential molecular mechanism of formononetin in PC-3 prostate cancer cells to further understand the pharmacological effects of formononetin and provide more references for intensive research. Materials and Methods: PC-3 cells were incubated with different doses of formononetin for 24 h or 48 h. After that, cell viability was measured by Cell Counting Kit-8, and apoptosis was analyzed by Hoechst 33258 stains. The expression levels of tumor-related factors such as long noncoding RNA (LncRNA) H19, Bax, and Bcl-2 were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot methods. Subsequently, PC-3 cells were infected with a lentiviral vector to overexpress or knock down H19, and then, the expression of insulin-like growth factor-1 receptor (IGF-1R) mRNA was measured by RT-qPCR. Results: Formononetin significantly inhibited the viability of PC-3 cells and promoted apoptosis in a time-dose-dependent manner. We observed that the expressions of lncRNA H19 and Bcl-2 were significantly downregulated compared with the untreated group, while an opposite pattern was observed for Bax. According to the results of gene interaction experiments, IGF-1R may be a downstream target of H19 in PC-3 cells. Conclusion: Our results present evidence that formononetin induced apoptosis of PC-3 cells by regulating lncRNA H19 and the mitochondrial apoptosis pathway. Furthermore, we put forward the hypothesis that formononetin has an interference effect on the H19/IGF-1R pathway, which remains to be further confirmed.