The objective of the study was to investigate the pharmacokinetics and efficacy of 5-FU entrapped pH-sensitive liposomal nanoparticles with surface-modified anti-epidermal growth factor receptor (EGFR) antibody (pHLNps-5-FU) delivery system. Cytotoxicity of 5-FU and pHLNps-5-FU was determined in vitro against HCT-116 cells. The biodistribution and pharmacokinetic parameters of the administered 5-FU and pHLNps-5-FU as well as efficacy of 5-FU and pHLNps-5-FU were determined in HCT-116 subcutaneous mouse model. Mean size of pHLNp-5-FU was 164.3 ± 8.4 nm with entrapment efficiency (E.E) of 54.17%. While cytotoxicity of 5-FU and pHLNps-5-FU showed a strong dose-dependent, pHLNps-5-FU proved to be more effective (2-3 fold high) than that of 5-FU against HCT-116 cells. Pharmacokinetic study showed a prolonged plasma circulation of pHLNps-5-FU and a more significant body exposure while accumulation of pHLNps-5-FU in tumor was significantly higher than that of free 5-FU. Further, the efficacy of pHLNps-5-FU, was greater than free 5-FU at equivalent 5-FU dose. The study suggests that pHLNps may be an effective drug delivery system to enhance the anticancer activity of 5-FU against colorectal tumor growth.