Ecdysterone is a naturally occurring steroid hormone secreted by arthropods, including insects, to regulate the molting process of larvae. Ecdysterone also has many pharmacological functions such as promoting cell growth and inducing human epidermal cell differentiation.Suspension culture of Ajuga lobata D. Don cells provides a method of synthesis of the phytoecdysteroid β-ecdysterone (β-EC). In this study, we tried to characterize the culture conditions to optimize β-EC production. Growth of A. lobata cells fit the logistic equation curve, with a growth cycle of 19 days and the stationary phase of 11 to 17 days. Medium conductivity was negatively correlated with dry cell weight and β-EC accumulation, thus could be used to determine the optimal time for cell harvest. Continuous subculture reduced β-EC synthesis, but supplementing medium with β-EC precursors mevalonic (MVA), metabolic inhibitors α-pinene and elicitor NO could significantly promote cell growth and influence β-EC accumulation. Combination of α-pinene, MVA and SNP significantly elevated β-EC accumulation, thus may synergistically enhance β-EC synthesis in A. lobata. The optimal concentrations of α-pinene, MVA and NO donor SNP in the suspension culture were 50 μL/L, 10 mg/L, 80 μmol/L, respectively.