Hydroxylation modification is crucial for maintaining the structural stability and biological activity of the collagen triple helix. To achieve a hydroxylation level in recombinant collagen produced in Escherichia coli comparable to its natural counterpart (12%~14%), highly hydroxylated recombinant human type III collagen (rhCOL3A1, 41.5 kDa) was prepared. This was accomplished using a previously constructed engineered E. coli strain capable of efficient rhCOL3A1 expression, combined with a strategy involving the exogenous addition of hydroxyproline (Hyp) and CaCl2. By varying the concentration of Hyp supplementation (0~200 mmol•L-1) in the culture medium, rhCOL3A1 products with different hydroxylation rates were obtained. After purification, high-performance liquid chromatography analysis indicated that the proline hydroxylation rate of rhCOL3A1 could reach 63.6%, with Hyp accounting for 13.9% of the total amino acid sequence, comparable to the hydroxylation level of natural collagen. Studies have shown that proline hydroxylation significantly enhances the structural stability and cellular activity of rhCOL3A1. Structural and functional validation of the hydroxylated rhCOL3A1 was performed. Circular dichroism spectroscopy confirmed the formation of a protein triple helix, and the hydroxylated form showed markedly increased resistance to trypsin degradation. Biological activity assays revealed that the hydroxylated rhCOL3A1 showed increases of 23.2% and 379.3% in in cell viability and cell adhesion capability, respectively, compared to the non-hydroxylated form. The technique established in this study provides a novel approach and theoretical foundation for the hydroxylation of recombinant collagen.