Papaver pseudo-orientale in section Oxytona of Papaver genus is an important source of alkaloids such as isothebaine. In this research, callus production and regeneration of this species from hypocotyl-cotyledon explants under in vitro conditions were investigated on Murashige and Skoog (MS) and Gamborg (B5) basic media supplemented with different levels of Kinetin (Kin), Benzyladenine (BA) and Naphthalene acetic acid (NAA) at 16-h photoperiod and 20 °C. Analysis of variance showed significant (P<0.01) differences between different culture media for callus induction rate, callus weight, shoot induction rate, shoot weight, root induction rate and root weight. Results showed that the proper media for P. pseudo-oriental callogenesis were MS medium supplemented with 0.5 mg/l BA and 0.5 mg/l NAA and B5 medium supplemented with 0.5 mg/l Kin and 1.0 mg/l NAA and the most favorable medium for regeneration of the studied species was B5 medium supplemented with 0.5 mg/l Kin and 0.5 mg/l NAA.