Low back pain is a leading health problem in the United States, which is most often resulted from nucleus pulposus (NP) degeneration. To date, the replacement of degenerated NP relies entirely on mechanical devices. However, a biological NP replacement implant is more desirable. Here, we report the regeneration of NP tissue using a biodegradable nanofibrous (NF) scaffold. Rabbit NP cells were seeded on the NF scaffolds to regenerate NP-like tissue both in vitro and in a subcutaneous implantation model. The NP cells on the NF scaffolds proliferated faster than those on control solid-walled (SW) scaffolds in vitro. Significantly more extracellular matrix (ECM) production (glycosaminoglycan and type II collagen) was found on the NF scaffolds than on the control SW scaffolds. The constructs were then implanted in the caudal spine of athymic rats for up to 12 weeks. The tissue-engineered NP could survive, produce functional ECM, remain in place, and maintain the disc height, which is similar to the native NP tissue.