Expanded polytetrafluoroethylene (ePTFE) is a suture material for annuloplasty in aortic valve repair. For this particular application, it should induce minimal local stress and promote rapid tissue incorporation. To achieve this, a novel ePTFE suture with a larger diameter and high porosity in its midsection has been developed. Herein, we analyzed the acute and chronic tissue reaction to this suture material compared with a commercially available control ePTFE suture. Novel and control suture samples were implanted into dorsal skinfold chambers of BALB/c mice to analyze the early inflammatory response using intravital fluorescence microscopy over 14 days. Additional suture samples were implanted for 4 and 12 weeks in the flank musculature of mice and analyzed by histology and immunohistochemistry. The implantation of novel and control ePTFE suture into the dorsal skinfold chamber did not induce an acute inflammation, as indicated by physiological numbers of rolling and adherent leukocytes in all analyzed venules. Chronic implantation into the flank musculature showed a better tissue incorporation of the novel ePTFE suture with more infiltrating cells and a higher content of Sirius red+ collagen fibers when compared with controls. Cell proliferation and viability as well as numbers of recruited CD68+ macrophages, myeloperoxidase+ neutrophilic granulocytes and CD3+ lymphocytes did not significantly differ between the groups. The novel ePTFE suture exhibits a good in vivo biocompatibility which is comparable to that of the control suture. Due to its improved tissue incorporation, it may provide a better long-term stability during annuloplasty.