Adult mammals have limited capacity to regenerate functional cells. Promisingly, in vivo transdifferentiation heralds the possibility of regeneration by lineage reprogramming from other fully differentiated cells. However, the process of regeneration by in vivo transdifferentiation in mammals is poorly understood. Using pancreatic β cell regeneration as a paradigm, we performed a single-cell transcriptomic study of in vivo transdifferentiation from adult mouse acinar cells to induced β cells. Using unsupervised clustering analysis and lineage trajectory construction, we uncovered that the cell fate remodeling trajectory was linear at the initial stage and the reprogrammed cells either evolved to induced β cells or toward a "dead-end" state after day 4.Moreover, functional analyses identified both p53 and Dnmt3a that acted as reprogramming barriers during the process of in vivo transdifferentiation. Collectively, we decipher a high-resolution roadmap of regeneration by in vivo transdifferentiation and provide a detailed molecular blueprint to facilitate mammalian regeneration.