Abstract: Background and Objective: Aquilaria rugosa L.C. Kiet and Keβler (Thymelaeaceae family) species is considered to be endangered in its original mountainous forest habitats. This study was conducted to establish a protocol of callus and cell suspension of A. rugosa. Materials and Methods: Three healthy plant parts of A. rugosa including leaves, stems and roots of in vitro 2 months old seedlings were used as explants for callus induction in Murashige and Skoog (MS) medium with 2.0% sucrose and 0.8% phytoagar at pH = 5.7. The growth regulators were tested at different concentrations (0.0-4.0 mg L–1) for Indole Butyric Acid (IBA), Indole Acetic Acid (IAA), a-naphthalene acetic acid (NAA), 6-benzylaminopurine (BAP) and 0.5-1.0 mg L–1 for BAP to find out which types of growth regulators were the most efficient for inducing callus of A. rugosa. Results: The research indicated that leaves, stems and roots of in vitro 2 months old seedlings were suitable for callus induction. The combination between auxin and cytokine was better than using single auxin in callus formation. Murashige and Skoog’s (MS) medium supplemented with 2 mg L–1 IBA, 1 mg L–1 BAP was the most efficient for inducing callus of A. rugosa (108.53±4.47 mg FW/leaf explant). The maximum growth rate of callus was observed in the 4th week of culture on the same maintenance media. Effective establishment of A. rugosa cell suspension was recorded with 2 g of initial callus fresh weight and in sucrose concentrations 40 g L–1. Conclusion: The research also suggested that medium supplemented with 2 mg L–1 IBA,1 mg L–1 BAP was the most suitable for inducing callus and initial inoculum sizes and sucrose concentration had effects on the growth of cells A. rugosa.