Background: Hyperoxaluria may result from increased endogenous production or overabsorption of dietary oxalate in the gastrointestinal tract leading to nephrolithiasis and, in some, to oxalate nephropathy and chronic kidney disease. ALLN-177 is an oral formulation of a recombinant, oxalate specific, microbial enzyme oxalate decarboxylase intended to treat secondary hyperoxaluria by degrading dietary oxalate in the gastrointestinal tract, thereby reducing its absorption and subsequent excretion in the urine. Methods: This double-blind, placebo controlled, randomized, cross-over, phase 1 study of ALLN-177 evaluated the tolerability of ALLN-177 and its effect on urinary oxalate excretion in 30 healthy volunteers with hyperoxaluria induced by ingestion of a high oxalate, low calcium (HOLC) diet. The primary end point was the difference in the mean 24-hour urinary oxalate excretion during the ALLN-177 treatment period compared with the placebo treatment period. Results: The daily urinary oxalate excretion increased in the study population from 27.2 ± 9.5 mg/day during screening to 80.8 ± 24.1 mg/day (mean ± SD) on the HOLC diet before introducing ALLN-177 or placebo therapy for 7 days. Compared to placebo, ALLN-177 treatment reduced urinary oxalate by 11.6 ± 2.7 mg/day, p = 0.0002 (least squares mean ± SD). Conclusions: In healthy volunteers, with diet-induced hyperoxaluria treatment with ALLN-177, when compared to placebo, significantly reduced urinary oxalate excretion by degrading dietary oxalate in the gastrointestinal tract and thereby reducing its absorption. ALLN-177 may represent a new approach for managing secondary hyperoxaluria and its complications.