Papilionthe hookeriana Rchb.f. (Orchidaceae), popularly known as ‘Anggrek Pinsil’ in Indonesia, is a perennial ephiphyte orchid, found only at Dendam Tak Sudah Lake in Bengkulu. The aims of this research were to find the best sterilization technique of P. hookeriana explant and to induce maximum formation of embryogenic calli. Rapid multiplication of this orchid was achieved through culture of shoot tips and young leaf segments of mature plants by in vitro cultured on Murashige and Skoog (MS) basal medium enriched with 50 g L-1 banana pulp. The experiment used Completely Randomized Design (CRD) with three replications. In the first stage, the explants were sterilized using three compositions of sterilant. In the second stage, the explants were planted on the MS basal medium with addition of five levels of 2,4-Diclhorophenoxy Acetis Acid concentrations, namely 0, 0.25, 0.50, 0.75, and 1.00 mg L-1. The result showed that the best sterilization method to reduce explant contaminant was method 3, in which the explant was washed with detergent, rinsed with flowing water, soaked in 0.1% (v/v) HgCl2 solution for 30 minutes, soaked again in the 10% (v/v) Natrium hypocloride solution for 20 minutes, rinsed three times with sterile water before planted and then soaked in sterile water + 10% (v/v) betadhine before planted on treated medium. This method was able to reduce contamination levels up to 70% from explants cultured for 5 months on MS medium. MS medium added with 1.00 mg L-1 2,4-D produced the highest number of embryogenic calli, and the biggest callus diameter ( 3.5 cm ), characterized by transparent green color and friable callus structure