|Chitosan nanoparticles are potential system for nasal vaccine delivery. The important factor that influences the efficacy of delivery system and immune response is size of particles. The objective of this research was to study of the optimal size of chitosan nanoparticles encapsulated with ovalbumin (OVA) as model antigen for nasal vaccine delivery. Chitosan nanoparticles were prepared by ionotropic gelation method with changing of chitosan concentrations from 1-3 mg/mL and the mass ratio of chitosan to sodium tripolyphosphate (TPP) from 3=1 to 5=1. Nine formulas were examined for physicochemical properties such as size and zeta potential of particles, entrapment efficiency, loading and releasing capacity by BCA Protein Assay, stability of antigen and nanoparticles by SDS-PAGE. Three formulas represented small (< 500 nm), medium (500 – 1,000 nm) and large size (1,000 – 5,000 nm) of chitosan nanoparticles encapsulated with ovalbumin were evaluated for in vivo immunostimulating activity related to ovalbumin solution by enzyme-linked immunosorbent assay (ELISA). 20 μL of each formula containing 20 μg of ovalbumin were intranasal given to Balb/c mice at the week of 0, 3, 6 and killed at the week of 9. Blood, feces, saliva, nasal lavage and vaginal lavage were selected. Humoral immune response (HIR) was evaluated from Immunoglobulin G (IgG) levels in serum as systemic immune response and secretory Immunoglobulin A (sIgA) levels in fecal extract, saliva, nasal lavage and vaginal lavage as mucosal immune response. Cell-mediated immune response (CMIR) was evaluated from splenocyteInterleukin-4 (IL-4) and Interferon-γ (IFN-γ). The results showed that formulas composed of 3 mg/mLchitosan were stable. When the mass ratio of chitosan to sodium tripolyphosphate (TPP) was increased from 3=1, 4=1 and 5=1,the sizes of chitosan nanoparticles were also increased from small (249.67 ± 29.6 nm), medium (559 ± 17.21 nm) and large size (1,240 ± 62.45 nm). But the percentages of entrapment efficiency (76.49 ± 4.72, 68.52 ± 6.01 and 66.77 7.95) and loading capacity (31.62 ± 1.52, 27.61 ± 1.68 and 27.03 ± 1.27) were decreased, respectively. The antigen releasing capacity from nanoparticles of these three formulas were all better at pH 4.5 than pH 6.8 and pH 7.4. The percentages of releasing capacity in pH 4.5 within 2 days were 86.62 ± 1.52, 85.20 ± 1.44 and 83.02± 1.36, respectively. After kept those lyophilized formulas at 4 °C for 3 months, they were still stable both antigen and chitosan nanoparticles. The result of immunostimulating activity exhibited that chitosan nanoparticles encapsulated with ovalbumin stimulated HIRbetter than ovalbumin solution. But there were no significantly difference between the particles sizes. However, the trends of sIgA levels in small and medium size were found to increase in saliva, nasal lavage and vaginal lavage. CMIR immunostimulating activity of chitosan nanoparticles encapsulated with ovalbumin was also more than ovalbumin solution.Small and medium size of nanoparticles had IL-4 and IFN-γ level more than large size. In conclusion, the optimal sizes of chitosan nanoparticles for nasal vaccine delivery are small and medium sizes. Because they had better physicochemical property such as positive surface charge, high entrapment efficiency, high releasing capacity especially at pH 4.5 and good stability both of antigen and delivery system. Moreover, they also stimulated HIR both mucosal immune responseandsystemic immune response and CMIR.