Adenosine A1 receptor (A1AR) signaling exerts important functions in the central nervous system (CNS). Among all CNS cell types, oligodendrocyte (OL) lineage cells possess the highest A1AR expression levels. However, in vivo data regarding the functions of A1ARs in OL lineage cells remain elusive. Here, we first generated NG2-CreERT2xA1ARfl/fl conditional knockout (cKO) mice to specifically delete A1ARs from oligodendrocyte precursor cells (OPCs) upon tamoxifen administration. Immunohistochemical analysis for OL lineage markers PDGFRα, APC CC1, as well as BrdU incorporation detected unaltered differentiation and proliferation of OPCs in the corpus callosum (CC) of A1AR cKO mice during development. Next, we established a modified mouse model of demyelination with a three-week cuprizone (CPZ) treatment, resulting in significant demyelination, glial activation as well as enhanced expression of ATP hydrolases converting extracellular ATP to adenosine. Pharmacological intervention experiments suggested adenosine signaling inhibits OL regeneration via A1ARs. Therefore, we applied the modified CPZ treatment protocol to the A1AR cKO mice to study the functions of OPC-specific A1ARs during de-/remyelination. After the withdrawal of cuprizone, we observed increased proliferation and differentiation of OPCs in the CC of A1AR cKO mice during de-/remyelination, leading to enhanced OL regeneration compared to control mice. Furthermore, we found transiently elevated microglia numbers at the end of CPZ treatment, suggesting microglial functions are involved in the mechanisms underlying the enhanced OL regeneration in cKO mice. Taken together, our results suggest A1ARs do not significantly affect the proliferation and differentiation of OPCs during development, but negatively regulate OL regeneration after CPZ-induced demyelination.