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The new folate-targeted amphiphilic cyclodextrin nanoparticles carrying chemotherapeutic drug PCX to breast tumors result in higher anticancer efficacy with reduced side effects and equivalent efficacy capable of cellular internalization with a folate dependent mechanism.As nanomedicines are gaining momentum in the therapy of cancer, new biomaterials emerge as alternative platforms for the delivery of anticancer drugs with bioavailability problems. In this study, two novel amphiphilic cyclodextrins (FCD-1 and FCD-2) conjugated with folate group to enable active targeting to folate positive breast tumors were introduced. The objective of this study was to develop and characterize new folated-CD nanoparticles via 32 factorial design for optimal final parameters. Full physicochemical characterization studies were performed. Blank and paclitaxel loaded FCD-1 and FCD-2 nanoparticles remained within the range of 70–275 nm and 125–185 nm, respectively. Zeta potential values were neutral and −20 mV for FCD-1 and FCD-2 nanoparticles, respectively. Drug release studies showed initial burst release followed by a longer sustained release. Blank nanoparticles had no cytotoxicity against L929 cells. T-47D and ZR-75-1 human breast cancer cells with different levels of folate receptor expression were used to assess anti-cancer efficacy. Through targeting the folate receptor, these nanoparticles were efficiently engulfed by the breast cancer cells. Additionally, breast cancer cells became more sensitive to cytotoxic and/or cytostatic effects of PCX delivered by FCD-1 and FCD-2. In conclusion, these novel folate-conjugated cyclodextrin nanoparticles can therefore be considered as promising alternative systems for safe and effective delivery of paclitaxel with a folate-dependent mechanism.