To investigate the response of cotton seedlings growth and proline metabolism to low- and excess-boron stress treatment conditions, the experiment was conducted using ‘E kang 10’ as experimental material using hydroponic method in a greenhouse of Huazhong Agricultural University. Boron (B) were applied at four levels at 0 mg L-1 (B0, no boron), 0.002 mg L-1 (B0.002, low concentration boron), 0.20 mg L-1 (B0.2, CK, sufficient concentration boron), and 50 mg L-1(B50, high concentration boron). The results showed that B0, B0.002, and B50 treatments significantly decreased the fresh and dry weights of plants, and inhibited root elongation relative to sufficient boron (CK, B0.2) treatment. With the increase of B concentration, B content in roots, stems, and leaves of cotton seedlings increased gradiently. Among them, B contents in the leaves under B0.2 and B50 treatments were higher than those in roots and stems, while B content in the roots was increased in leaves and stems under B0 and B0.002 treatments. Under low- and high-boron stress treatments, the content of proline in leaves increased dramatically, while proline in roots decreased. Further analysis of related enzyme activities in proline metabolism, we found that B0.002 and B50 treatments promoted the activities of Δ1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine-δ-aminotransferase (OAT) in leaves, but decreased the activity of proline dehydrogenase (ProDH) compared to CK; and the activity of Δ1-pyrroline-5- carboxylate reductase (P5CR) in leaves under B50 treatment was obviously increased, but there was no significant difference under B0.002 treatment. In addition, compared with CK, B50 treatment reduced the enzyme activities of OAT and P5CR in roots, while B0.002 treatment prominently increased the activities of P5CS and ProDH. The results showed that both low- and excess-boron stress inhibited the growth of cotton seedlings. Under boron stress, proline mainly was accumulated in the leaves of cotton seedlings, and the content of proline in roots decreased significantly. In the case of boron deficiency and boron toxicity, the accumulation of proline in leaves was mainly through regulating the activities of key enzymes (OAT, P5CS synthetase, and ProDH degrading enzyme) in proline Glu and Orn pathways, resulting in the proline synthesis rate higher than its degradation rate. However, in roots, the proline content was decreased mainly by promoting the degradation of proline under boron deficiency stress. Under high boron stress, proline synthesis and decomposition were inhibited mainly by reducing the activities of OAT, P5CS synthetase and ProDH decomposing enzyme, but the inhibitory effect on proline synthesis was much greater than its degradation, which eventually led to the decrease of proline content in roots.