Abstract: Background and Objective: Pepper (Capsicum annuum L.) is one of the major vegetable and spice crops grown worldwide. This study highlighted the economic and importance process for in vitro capsaicin production from pepper plant. As well as to study the effect of MS medium supplemented with 2,4-D in combinations with 0.2 mg L–1 Kin. for callus and suspension production was implemented. As well as, the influence of A. niger as biotic and methyl jasmonate as abiotic elicitors on capsaicin accumulation was investigated. Materials and Methods: For this purpose, callus cultures were prepared from seeds of pepper and subcultured. After 28 days, calli formation (%), fresh weight (g/Jar) and dry weight (g/Jar) was calculated. Cell number and packed cell volume were calculated from their cell suspension. In the end, capsaicin was extracted and colorimetrically quantified. Results: The highest percentage of calli formation was recorded with hypocotyl, leaf and root explants, respectively. The MS medium fortified with 3.0 mg L–1 2,4-D+0.2 mg L–1 Kin. In addition, the maximum P.C.V was recorded after 16 days of cultivation on the same medium. Furthermore, it was found that fortified MS medium with 1.5% of A. niger in combination with 100 μM of methyl jasmonate achieved of cell growth parameters and capsaicin accumulation in significant rate during 16 days of cultivation. Conclusion: Fortified of MS medium with 3.0 mg L–1 2,4-D+0.2 mg L–1 Kin. showed the optimized medium for both callus and suspension production. Moreover, augmentation of MS medium with 1.5% of A. niger in combination with 100 μM of methyl jasmonate enhanced of capsaicin accumulation in significant rate during 16 days of cultivation.