The effects of Timentin and cefotaxime (Cef) on shoot regeneration of the London plane tree (Platanus acerifolia Willd.) and their use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were compared. Shoot regeneration was significantly reduced on the media with Cef at concentrations from 100 to 500 mg·L–1. Timentin showed negative effect on plant regeneration at concentrations of 100 and 500 mg·L–1; however, 300 mg·L–1 Timentin was shown to facilitate shoot regeneration significantly and the regeneration frequency increased from 64% (control) to 88%. Effective suppression of A. tumefaciens could be obtained with 500 mg·L–1 Cef, but plant regeneration was completely inhibited at this level. The A. tumefaciens on infected P. acerifolia leaf tissues was visually undetectable after three subcultures on a medium with 300 mg·L–1 Timentin. Con-sidering the effect of Cef and Timentin on plant regeneration and suppression of Agrobacteria, Timentin at 300 mg·L–1 is the pre-ferred application in A. tumefaciens-mediated transformation of P. acerifolia.