Silica nanoparticles (NPs) with various surface properties were introduced in poly(3-hydroxybutyrate) (PHB) matrix and the their effect on the dispersion, crystallization behavior, and reinforcement in the nanocomposites was discussed in this article. Two kinds of commercial fumed silica NPs and two kinds of self-prepared sol-gel silica (bare and PEGylated) NPs were used. The cross-sectional SEM (scanning electron microscopy) images, provided the micrometer scale view (observation area: 12.6×8.2 μm2), showed that commercial fumed silica and PEG–silica NPs were aggregated and well-dispersed in PHB matrix, respectively. Similarly, Morisita’s analysis of TEM (transmission electron microscopy) images (observation area: 2.4×1.6 μm2) indicated that PEG-silica NPs were Poisson dispersed and commercial fumed silica NPs were serious aggregated in PHB matrix. However, SEM-EDX (energy dispersive X-ray analysis) Si-mapping micrographs, provided the millimeter scale view (observation area: 0.79×0.61 mm2), showed that four kinds of silica NPs were well-dispersed in PHB matrix. PLM (polarized light microscopy) images indicated that spherulite growth rate and morphology of PHB did not change obviously upon the addition of various silica NPs, except the PHB/PEG–silica system. PHB/PEG–silica showed a decreased spherulite growth rate, which was consistent with the DSC (differential scanning calorimetry) results, because the good miscibility between PHB and the grafted PEG chains on PEG–silica could decrease the polymer chain mobility during crystallization. The Young’s modulus and tensile strength of the PHB were enhanced by up to 34% and 63% by adding a small amount of PEG–silica. Fully well-dispersed PEG–silica NPs functioned as physical cross-linking centers for enhancing the mechanical properties of PHB but as retarding agents for reducing the crystallization rate.