Introduction: The creation of synthetic reservoirs for bladder replacement has been limited by challenges of interfacing synthetic materials and native tissue. We sought to overcome this challenge by utilizing a novel bilayer silk fibroin scaffold (BLSF) as an intermediary toward the development of an acellular prosthetic reservoir. Methods: Under institutionally approved protocols, 3D-printed reservoirs were implanted in six juvenile female pigs after cystectomy. BLSF was attached to the in situ prosthetic reservoir serving as an intermediary to native ureteral and urethral tissue anastomoses. Our first protocol allowed four pigs to be survived up to 7 days, and the second protocol allowed two pigs to be survived for up to 1 year. At the first sign of functional decline or the end of the study period, the animals were euthanized, and kidneys, ureters, prosthetic bladder, and urethra were harvested en bloc for histopathology analysis. Results: The first two pigs had anastomotic urine leaks because of design flaws resulting in early termination. The third pig had acute renal failure resulting in early termination. The artificial bladder design was modified in subsequent iterations. The fourth pig survived for 7 days and, upon autopsy, had intact urethral and ureteral anastomoses. The fifth and sixth pigs survived for 11 and 12 weeks, respectively, before they were sacrificed because of failure to thrive. One animal developed an enteric fistula. The other animal had an intact anastomosis, and the BLFS was identified at the ureteral and urethral anastomoses on histopathologic analysis. Conclusions: Replacing the porcine bladder with a prosthetic bladder was achieved for up to 3 months, the second longest survival period for a nonbiologic bladder alternative. BLSF was used for the first time to create an interface between synthetic material and biologic tissue by allowing ingrowth of urothelium onto the acellular alloplastic bladder.