Conjugation of growth factors to a carrier is a favorable method to improve their efficacy as therapeutic molecules. Here, we report the carrier size effect on bioactivity of human epidermal growth factor (hEGF) conjugated to polystyrene particles. BALB/3T3 cells were treated with hEGF-conjugated particles (hEGF-conjs) sized from 20 to 1000 nm. At hEGF concentrations less than 0.5 ng ml-1, free hEGF was more potent than the hEGF-conjs at inducing cell proliferation. However, cell proliferation was size-dependent at higher concentrations of hEGF i.e. hEGF-conjs sized equal to or less than 200 nm displayed lower cell proliferation, compared to free hEGF, but larger particles showed increased cell proliferation. This is in agreement with previous studies showing accumulation of activated-EGFRs in early endosomes triggers apoptosis of A431 and HeLa cells. The confocal microscopy and co-localization fluorescence staining showed the 500 and 1000 nm hEGF-conjs exclusively remained on the cell surface, probably enabling them to activate EGF receptors for a longer time. Conversely, smaller particles were mostly inside the cells, indicating their rapid endocytosis. Similarly, A431 cells treated with 20 nm hEGF-conj, endocytosed the particles and experienced decreased cell proliferation, while the 500 and 1000 nm hEGF-conjs were not internalized, and induced partial cell proliferation. Moreover, we showed multivalency of hEGF-conjs is not the cause of enhanced cell proliferation by large particles, as the degree of EGFR phosphorylation by free EGF was higher, compared to hEGF-conjs. Our results suggest the potential of micron-sized particles as a carrier for hEGF to enhance cell proliferation, which could be explored as a promising approach for topical application of growth factors for accelerating wound healing.