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Purpose: Adaptive growth of the intestinal mucosa in response to massive gut loss is critical for autonomy from parenteral nutrition. Normal intestinal adaptation is characterized by the structural changes of increases in villus height and crypt depth. Our laboratory has previously demonstrated that p21-null mice do not to adapt after small bowel resection (SBR). Further studies reveal that these animals have dramatically increased levels of Rb protein in the crypt cells. Alternatively, we have previously shown that intestine-specific Rb deficiency results in intestinal mucosal hyperplasia manifested by increased villus height, crypt depth, and crypt cell proliferative rate. In this study, we tested the hypothesis that the magnitude of Rb expression in p21-null mice is crucial for the normal adaptive response to massive SBR. Methods: To reduce Rb protein levels in p21-null mice, one Rb allele was deleted by breeding villin Cre Rb (flox/flox) mice with p21-null mice, resulting in offspring genotyped as Villin Cre (+); Rb (+/flox); p21 (-/-). (These mice will be referred to as p21-null-Rb-hets). All genotypes were confirmed by PCR. Enterocyte crypt fractions were isolated and Rb protein levels were evaluated by Western Blot and quantified by optical density using Image-J software. Mice were subjected to either a 50% small bowel resection or sham (transection and reanastomosis) operation. The remnant ileum was harvested 7 days after operation, and adaptation was evaluated by measuring villus height and crypt depth. Results: Elevated crypt Rb levels in p21-null mice were successfully reduced to levels comparable to wild-type animals by deleting one Rb allele. Unlike the p21-null mice, the p21-null-Rb-het mice that underwent SBR did manifest adaptive changes in crypt depth and villus height when compared to sham animals. (crypts: Sham 59.6+2.3 um, SBR 74.3+0.4 um, p<0.001; villus: Sham 167.5+0.8 um, SBR 256.5+¬17.5 um, p>0.006). Conclusion: By deleting one Rb allele in a p21-null background, Rb levels were successfully decreased to levels comparable to wild-type animals. Correspondingly, the normal adaptive response to intestinal resection was restored in these p21 null mice. These findings suggest a crucial mechanistic role for Rb in p21-directed small intestine adaptation responses to massive small bowel resection.