The effects of various concentrations of toad bufanolide diene (BD) on the growth of Tetrahymena thermophila BF5 were investigated by means of a thermal activity monitor (TAM) system. The extent and duration of toxic effects on metabolismwere evaluated by studying the growth rate constant (k), maximumheat-output power (Pmax) and half inhibitory concentration (IC50). Experimental results showed that the values of k and Pmax decreased as the concentrations of BDincreased. Furthermore, the growth rate constant (k) was linked to the concentration of BD: rcinobufagin=0.9910 (IC50=119.2 μg·mL-1); rbufalin=0.9923 (IC50=106.14 μg·mL-1); rbufotalin=0.9977 (IC50=73.80 μg·mL-1); rgamabufotalin=0.9923 (IC50=54.75 μg·mL-1). The sequence of toxicity for the four BDs was: gamabufotalin>bufotalin>bufalin>cinobufagin. The α-hydroxy group at C11, β-hydroxy group at C14, β-acetoxy at C16 and the non-dehydrolysis between C14 and C15 can possibly improve the toxicity of BD towards Tetrahymena thermophila BF5.