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Calothrixin B, a marine-derived compound, displays a broad spectrum of biological properties including anticancer effects. Calothrixin B has low water solubility, meaning that its therapeutic application is limited. In the present study, we explored the delivery of calothrixin B in the form of liposomal nanoparticles for its anticancer activity. Liposomes were prepared with cholesterol, and either DOPC or both DOPC and DOPE. Calothrixin B liposomes exhibited small sizes, ranging from 108 to 117 nm, and negative surface charges. High encapsulation efficiencies of calothrixin B (>89%) were achieved in all formulations. Liposomes composed of DOPC, DOPE and cholesterol resulted in the release of 87% of calothrixin B within 48 h, while a slower release rate was observed for the liposome without DOPE, with 56% release of calothrixin B. The cytotoxicity of calothrixin B in DOPC, DOPE and cholesterol liposomes was more pronounced than that in liposomes without DOPE. In comparison with DMSO-calothrixin B, calothrixin B in DOPC, DOPE and cholesterol liposomes showed significantly increased toxicity against A549 and comparable activity against MCF-7. Confocal microscopy confirmed that DMSO-calothrixin B and calothrixin B liposomes could induce cell death by the same mechanism. Calothrixin B liposomes demonstrate significant potential as delivery carriers for cancer therapy.