Using pharmacological and biochemical approaches, Ca2+ involved in the betulin production in mycelia of Phellinus linteus induced by hydrogen sulfide (H2S) were investigated. The results showed that 2 mM H2S donor NaHS or 10 mM CaCl2 was found to enhance the betulin content in the mycelia of Phellinus to the maximum, which were 112.43 and 93.24 % higher than that in the control, respectively. Further, NaHS and CaCl2 co-treatment also showed positive outcome, which were 128.95 or 24.52 % higher than that in the control or NaHS treatment. At the same time, NaHS also enhanced the content of Ca2+ and CaM. But, the above positive inductive effects for Ca2+, CaM, and betulin production can be blocked with either Ca2+ channel blocker (LaCl3, 2-aminoethoxydiphenyl borate) or Ca2+ chelator (ethylenediaminetetraacetic acid (EDTA)). Among of them, betulin content was reduced 35.06 % by NaHS and EGTA to the minimum, and this reduction could be reversed by the application of CaCl2 (NaHS + EGTA + CaCl2). From above results, it can be concluded that endogenous and exogenous calcium involved in the betulin production from submerged culture of P. linteus induced by hydrogen sulfide.