Ordered mesoporous carbon is a kind of novel carrier for intracellular drug release. There are few reports on the use of mesoporous carbon nanospheres (MCNs) as the transmembrane deliverer in human cancer cells; on the other hand, the particle size of MCNs synthesized by hard templates is usually larger than 100 nm. It is accepted that the optimal size of a transmembrane delivery vehicle should be less than 100 nm in diameter and the surface should be hydrophilic to circumvent clearance by macrophages, to maximize circulation times and targeting ability. In this work, MCNs with a diameter of ca. 90 nm have been developed as a targeted drug delivery system of an anticancer drug, doxorubicin (DOX). The small MCNs were synthesized using triblock copolymer Pluronic F127 as a template. The MCNs were first treated by acid to improve its dispersion property in an aqueous solution, and then modified by folic acid through EDC-NHS. The structure of the MCNs was well characterized by transmission electron microscopy, small-angle X-ray scattering, nitrogen adsorption/desorption and dynamic light scattering. pH-dependent drug release is successfully achieved due to the supramolecular π-π stacking between DOX and the carbonaceous structures. By effective passive and active targeting, MCNs can be readily internalized into HeLa cells, where the carried DOX can be efficiently released in the acidic microenvironment of the tumors for further therapy. The results from confocal laser scanning microscope and flow cytometry demonstrated that the cellular uptake efficiency of MCNs toward HeLa cells was increased through the functionalization with folic acid, and the folate modified MCNs show much higher endocytosis properties toward HeLa cells (folate receptor positive) than toward MCF-7 cells (folate receptor negative). The cytotoxicities toward HeLa cells were studied by MTT method, which indicated that the cytotoxicities of DOX loaded mesoporous carbon nanoparticles was also enhanced due to the introduction of folic acid and targeted delivery, while the cytotoxicities of MCNs show very good biocompatibility toward both HeLa and KB cells.