We fabricated a highly efficient (with a solar-to-electricity conversion efficiency (η) of 5.51%), plastic-based, flexible dye-sensitized solar cell (DSSC). The photoanode was made of a crystalline mesoporous TiO2 film using a low-temperature electrophoretic deposition (EPD) process and compression treatment. The crystalline mesoporous TiO2 film was composed of secondary mesoporous TiO2 nanoparticles (MTNs, ca. 260 nm in size), synthesized by an aggregation of primary TiO2 nanocrystallites. In contrast to commercial TiO2 nanoparticles (i.e., P90, ca. 15.9 nm in size) that are widely used in DSSCs, the synthesized MTN-based film exhibited a higher surface area and porosity that increased dye adsorption, promoted effective electron transport, and enhanced light scattering, as evidenced by analysis of reflectance and absorbance spectra, X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). We also optimized the compression pressure for MTN- and P90-based DSSCs in order to achieve maximum efficiency. We further systematically investigated the cause for the enhanced conversion efficiency of MTN-based DSSCs by measuring incident photon-to-electron conversion efficiency (IPCE) curves and electrochemical impedance spectra (EIS). IPCE results explained the increase in the short-circuit photocurrent density (JSC) for MTN-based DSSCs, and EIS results indicated that MTN-based DSSCs exhibited a larger diffusion coefficient (Deff), longer effective diffusion length (Ln), longer electron lifetime (τe), and lower charge transfer resistance (Rk), resulting in a higher power conversion efficiency. The MTN-based DSSC fabricated in the study showed great potential for application in plastic-based DSSCs using room temperature procedures.