Purpose <p>Antimicrobial peptides (AMPs), produced constitutively by all living organisms serve as critical components of the innate immune system. Owing to their broad-spectrum antimicrobial activity and low propensity for resistance development, AMPs represent promising alternatives to conventional antibiotics. Mesenchymal stem cells (MSCs), beyond their regenerative capabilities; have gained attention for their innate antimicrobial properties. In this study, we performed de novo transcriptome analysis for the expression of AMPs in canine MSCs following bacterial priming and assessed the in vitro antimicrobial activity of the conditioned media.</p> Methods <p>MSCs were isolated and characterized from canine umbilical cord tissue. The cells were primed with <i>Staphylococcus aureus</i> by transwell culture method for 6&#xa0;h. The RNA was extracted from these primed cells and whole transcriptome profile was generated through next-generation RNA sequencing. The antimicrobial activity of primed-cell culture supernatant was assessed in vitro.</p> Results <p>Using Illumina Novaseq sequencer, we obtained 26.23&#xa0;million high quality clean reads and subsequently de novo assembled into 59,214 contigs which were annotated using Trinity, TransDecoder, UniProt database and validated with CAMPR4 database. The database could identify a total 30 potential AMPs; among them 8 were found which matched with the existing database and the remaining 22 were predicted to be AMPs. The conditioned media exhibited strong antibacterial effects against <i>S. aureus</i>, likely attributable to the secreted AMPs.</p> Conclusion <p>These findings highlight the capacity of canine MSCs to serve as a source of both known and novel AMPs that could be screened for prospective biotherapeutic strategies.</p> Graphical Abstract <p>Workflow of transcriptome analysis for AMP detection of cUC-MSC</p> <p></p>