Inotodiol, one tetracyclic triterpenoid isolated from inonotus obliquus (Chaga mushroom) possess excellent anticancer, antioxidant and anti-inflammatory activities, however it has not been applied to medical use due to its low solubility and low bioavailability. Liposome as a good nanodrug carrier with EPR and good biocompatibility has received much research attention. To improve the solubility and bioavailability of IOP, we prepared inotodiol liposomes (IOP-Lps) and folic-acid targeting IOP liposome (FA-IOP-Lps) by ultrasonic method, and their particle size, morphology, zeta potential, entrapment efficiency (EE) and drug loading rate (DL) were characterized by DLS, TEM, FT-IR and HPLC respectively. Their in vitro cytotoxicity of human cervical cancer cells HeLa, human liver cancer cells HepG2 and human breast cancer cells MCF-7 were assessed using the MTT assay. The results showed that IOP-Lps and FA-IOP-Lps possess significant anticancer effects. The results of TEM, FT-IR and DLS confirmed the formation of liposome. The particle size of IOP-Lps were 201.07±6.47 nm and ζ-potential of -50.2±0.5 mV, FA-IOP-Lps 224.33±1.86 nm and ζ-potential of -51.2±0.3 mV, the EE of IOP-Lps and FA-IOP-Lps were 79.14% and 77.33%, respectively. IOP-Lps and FA-IOP-Lps could selectively kill HeLa, HepG2 and MCF-7 cancer cells while nontoxic to normal L02 cells. This is the first study to give out in vitro information of inotodiol’ s anticancer effects based on nanocarriers. Besides, this drug delivery system is of good sustained release and targeted delivery effects to promote the utilization of both inotodiol and other natural hydrophobic compounds in target treatment of cancers.