To evaluate the effect of auxin and auxin analogues on in vitro rooting, and provide a useful suggestion for the establishment of efficient rooting protocol in plants, the roles of IAA, Indole-3-butyric acid (IBA), and α-naphthaleneacetic acid (NAA) in regulating root system development during the in vitro successive subculturing of mature trees in Pinus massoniana were studied. Firstly, results for variations in endogenous plant growth regulators (PGRs) levels and rooting capacity of shoots in vitro cultured on half-strength Murashige and Skoog medium (MMS) containing 1.2 µM l^-1 NAA showed that the endogenous IAA level in shoots was significantly enhanced with the increase of number of subcultures, and there was a remarkably linear relationship between the IAA level and rooting (%) or root number. The culture of shoots on half-strength MMS medium enriched with 4 µM l^-1 IAA resulted in increases in both rooting (%) and root number without any negative effect on root development, while 4 µM l^-1 IBA caused the decrease in root number. IBA of lower concentration (2 µM l^-1) enhanced rooting (%) and root number, but its promotive effects on root formation were delayed compared with effects of 4 µM l^-1 IAA. These results suggest that the use of IAA for the in vitro rooting of mature P. massoniana trees is efficient.