The lipid membrane composition defines the physical and pharmacological characteristics of liposomal drugs, and it can be tailored to meet the desired drug delivery needs. The current study is aimed to provide a sharper understanding of the lipid composition effect on doxorubicin (DOX) delivery kinetics, using cholesterol and phosphatidylcholine lipids (PCs) with different acyl chains in liposomal DOX formulations. The PCs were distearoyl (DSPC), dipalmitoyl (DPPC), dimyristoyl (DMPC) and egg-derived PC (EPC), either alone or in combination with cholesterol. Several characteristics were monitored, including DOX loading capacity of liposomes, DOX release in phosphate buffered saline (PBS), PBS/human plasma including buffy coat and human blood, cell uptake, as well as in vivo distribution and therapeutic effects in BALB/c mice bearing C26 colon carcinoma. Addition of cholesterol to liposomal formulation enhanced the particle size stability of the liposomes and the DOX-to-lipid ratio. EPC-liposomes and EPC/Cholesterol-liposomes showed few distinctive features. Overall, cholesterol decreased DOX release from the liposomes, and longer saturated fatty acyl chains in PC decreased DOX release and side-effects and increased the anti-tumor effects of liposomal DOX.