Polygonatum kingianum is usually used as a Chinese traditional homology of medicine and foods. P. kingianum polysaccharides exhibit various biological activities. To date, the processing of P. kingianum with Lactobacillus fermentation has been rarely explored. This study investigated the optimum fermentation conditions of P. kingianum using Lactobacillus plantarum and Lactobacillus rhamnosus by response surface methodology (RSM), followed by the extraction and characterization of fermented polysaccharides (namely MFP and SFP respectively), in contrast to non-fermented polysaccharide (NFP). Their changes in physicochemical properties, antioxidant, and cardioprotective activities were studied. High-performance size-exclusion chromatography analysis results demonstrated that a reduction in the proportion of the lower molecular weight fraction II (2.9 kD) of MFP and SFP. Compared to NFP, both MFP and SFP showed increased levels of galactose, galacturonic acid, and rhamnose, but decreased mannose, galactosamine, glucuronic acid, glucose, fucose, and fructose. Furthermore, the 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays revealed the higher antioxidant activities of MFP and SFP. Bioassay results showed that MFP and SFP possessed cardioprotective activities against hypoxia/reoxygenation injury in cardiomyocytes through the alleviation of oxidative stress and the inhibition of cell apoptosis. Collectively, these results could provide a reference for largescale production of polysaccharides form P. kingianum using Lactobacillus in medical and food industries. Display omitted •Process for the fermentation of Polygonatum kingianum was optimized.•Lactobacillus fermentation improved antioxidant activity of Polygonatum kingianum.•Fermentation altered the physicochemical properties of polysaccharides.•Fermented-polysaccharides exhibited superior cardioprotective potential.