Prostate cancer remains a major global health challenge, driving the need for innovative therapies. Nisin, an antimicrobial peptide from Lactobacillus lactis, has shown anticancer effects in various malignancies, yet its impact on prostate cancer and the prostate cancer antigen 3 (PCA3) long non-coding RNA (lncRNA) remains unstudied. This research aimed to investigate nisin's anticancer properties in prostate cancer cells, focusing on PCA3 lncRNA, apoptosis, and cell cycle pathways. Human prostate adenocarcinoma (LNCaP) and normal human foreskin fibroblast (HFF2) cells were treated with nisin. Cell viability was measured using MTT assays, while apoptosis and cell cycle progression were assessed via flow cytometry. Quantitative PCR (qPCR) evaluated gene expression of PCA3 lncRNA, apoptosis-related genes, cell cycle regulators, and PCA3-associated microRNAs and mRNAs. In-silico analysis of TCGA-PRAD data explored PCA3's regulatory network. Nisin selectively reduced LNCaP cell viability (IC50: 370.7 mu M at 24 h, 177.2 mu M at 48 h) compared to HFF2 cells (IC50: 887.8 mu M at 24 h, 406.5 mu M at 48 h). It induced time-dependent apoptosis and G1 phase cell cycle arrest in LNCaP cells. Nisin downregulated PCA3 lncRNA expression, upregulated miR-132-3p and miR-1261, and altered SREBP1 and PRKD3 gene expression, modulating the PCA3 regulatory network. This study is the first to explore nisin's anticancer effects in prostate cancer, uniquely targeting PCA3 lncRNA and its downstream regulatory pathways. Nisin demonstrates potent anticancer effects in prostate cancer cells by inducing apoptosis, arresting cell cycle progression, and modulating the PCA3 lncRNA network, suggesting its potential as a novel therapeutic agent.