Background Inactive human cathelicidin antimicrobial peptide is present in neutrophils, and proteinase 3 activates this peptide by producing active LL-37 peptide. LL-37 acts as a defensive peptide in the oral tissues. In the present study, the aim was to evaluate LL-37 and proteinase 3 levels in peri-implant sulcus fluid (PISF) in implants with and without peri-implantitis. Methods Patients who simultaneously had dental implants with peri-implantitis and without peri-implantitis were included in the study. Forty-four samples with peri-implantitis and 34 samples without peri-implantitis from 16 patients were obtained. Intraoral evaluations such as pocket depth, modified sulcus bleeding index, and modified plaque index were noted. Enzyme-linked immunosorbent assay was used for the evaluation of PISF LL-37 and proteinase 3 levels. Results PISF volume was significantly increased in the implants with peri-implantitis than those without peri-implantitis (p< 0.05). No difference was present between PISF LL-37 and proteinase 3 total amounts between the implants with and without peri-implantitis (p> 0.05). Pocket depths and PISF LL-37 and proteinase 3 levels were not correlated in the groups (p> 0.05). Conclusions PISF volume might be increased in response to peri-implant bone destruction. However, peri-implant tissue destruction caused by peri-implantitis does not seem to affect PISF LL-37 and proteinase 3 levels.