Leishmaniasis is caused by protozoan of Leishmania parasite. The pentavalent antimonials compounds remain the first-line treatment. J-binding protein encoded by JBP1 involves in starting synthesis of J base that is unique in kinetoplastida. In this study, we were assessed the JBP1 gene expression in exposure of different doses of glucantime. L. major (MRHO/IR/75/ER) promastigotes were distributed in groups for exposure with glucantime with end concentrations of 5, 10, 15, and 20 mg/ml. Then, RNA was extracted and cDNA synthesis was performed. Gene expression of JBP1 was assessed using SYBR Green Real Time PCR by ΔΔCT analysis. Gene expression of all groups exposure with different concentrations of glucantime were the same but the JBP1 gene expression showed 1.4 fold decreasing in groups with promastigotes exposed with glucantime with the end concentration of 5 mg/ml. The base J is synthesized with JBP1. More synthesis of J base is resulted in decreasing RNA polymerase II that could affect the gene expression of other genes. In this study, we showed that in high concentrations of glucantime, the JBP1 was increased resulted in increasing in J base synthesis. Therefore, it seems that expression regulation of the genes involving in drug exposure would be in the other mechanisms.