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AIM The aim of this study was to develop nanofiber-based sutures capable of inducing endogenous antimicrobial peptide production. METHODS We used co-axial electrospinning deposition and rolling to fabricate sutures containing pam3CSK4 peptide and 25-hydroxyvitamin D3 (25D3). RESULTS The diameters and mechanical properties of the sutures were adjustable to meet the criteria of United States Pharmacopeia designation. 25D3 exhibited a sustained release from nanofiber sutures over 4 weeks. Pam3CSK4 peptide also showed an initial burst followed by a sustained release over 4 weeks. The co-delivery of 25D3 and pam3CSK4 peptide enhanced cathelicidin antimicrobial peptide production from U937 cells and keratinocytes compared with 25D3 delivery alone. In addition, the 25D3/pam3CSK4 peptide co-loaded nanofiber sutures did not significantly influence proliferation of keratinocytes, fibroblasts, or the monocytic cell lines U937 and HL-60. CONCLUSION The use of 25D3/pam3CSK4 peptide co-loaded nanofiber sutures could potentially induce endogenous antimicrobial peptide production and reduce surgical site infections.