Lysine lactylation (Kla) is a recently identified post-translational modification derived from lactate that regulates diverse biological processes. Although Kla has been studied in several cancers, its role in prostate cancer (PC) remains unclear. The objective of this study is to profile Kla in PC in order to explore the mechanisms involved in PC progression.We performed global Kla profiling in PC-3M prostate cancer cells using affinity enrichment with anti-Kla antibodies, followed by LC-MS/MS. Bioinformatics analyses were conducted to explore the functional roles of Kla-modified proteins.We identified 681 Kla sites across 379 proteins, with modifications predominantly located in nuclear and cytoplasmic proteins. Enrichment analysis indicated Kla involvement in mRNA splicing, chromatin organization, and glycolysis/gluconeogenesis. Several multifunctional proteins, including AHNAK and nucleolin (NCL) harbor multiple Kla sites. Motif analysis indicated conserved amino acid patterns surrounding Kla sites. Notably, PC-3M cells showed reduced expression of sirtuin (SIRT)3, SIRT5, and SIRT6, which may underlie elevated Kla levels.This study presents the first comprehensive Kla landscape in PCa, suggesting its potential regulatory role in tumor progression. These findings provide a valuable resource for future studies and support Kla as a possible target for therapeutic intervention in prostate cancer.