High-protein diets often lead to an increase in urea concentration in follicular fluid of dairy cows, which may reduce oocyte competence. In the present study, maturation media were supplemented with urea (0, 20, 40 mg/dl), and amino acids (AAs) turnover was evaluated in the 24-h spent media of specimens (cell types), bovine cumulus-oocyte complexes (COCs), cumulus cells (CCs), or denuded oocytes (DOs). The main effects of urea and cell type, and their interaction were significant on the individual turnover (expect threonine, glycine, and tyrosine) and total turnover, depletion, and appearance of AAs. The results showed a high level of urea and DOs increased the depletion of all AAs and that of essential and non-AAs, respectively. Sensitivity analysis revealed the highest sensitivity of isoleucine, lysine, and tryptophan to urea, especially in DOs. Principal component analysis (PCA) evaluated the strong correlations between the turnover of: (1) glutamine, aspartic acid or glycine, and developmental competence and fertilization of COCs; (2) serine, isoleucine, valine or glutamic acid, and cleavage rate of DOs; and (3) serine, glutamine, aspartic acid or alanine, and CCs viability. In conclusion, urea significantly changed the turnover of AAs by COCs, CCs and DOs, and reduced the subsequent developmental competence of bovine oocytes.