The two-spotted spider mite, Tetranychus urticae Koch is the most harmful pest among many cropping systems, particularly vegetables and other annual crops. In this study, the methanol extract from the inner bark of Tabebuia impetiginosa was evaluated for acaricidal activity against T. urticae by using a leaf disk method and were compared with commercial acaricide, abamectin. A crude methanol extract of T. impetiginosa had strongly acaricidal activity at 2,000 ppm against the T. urticae. Methanol extract of T. impetiginosa was partitioned with hexane, chloroform, ethyl acetate, butanol and water fraction, successively. In this result, the chloroform fraction showed a strong acaricidal activity at 1,000 ppm. Therefore, active fraction of T. impetiginosa extract was purified by using silica gel column chromatography and high performance liquid chromatography. The structure of acaricidal component was analyzed by EI-MS, 1H-NMR and 13C-NMR spectra, and was identified as TI-3123. Based on the LC50 values of TI-3123 and abamectin against T. urticae were resulted in 0.011 mg/l and 0.340 mg/l, respectively. These results showed that acaricidal activity of the T. impetiginosa can be mostly attributed to TI-3123. Furthermore, TI-3123 was approximately 30.91 times more toxic than abamectin against T. urticae. All these results suggested that active component in T. impetiginosa derived materials could be use for biological control for T. urticae. Further studies should be performed to the structure activity relationship of TI-3123 and compared with its derivatives.