The association between intestinal permeability and sarcopenia remains unclear, and few studies have mentioned the relationship between intestinal permeability and skeletal muscle strength. The present cross-sectional community study was conducted in a rural area of Beijing to explore the association between intestinal permeability and handgrip strength (HGS) in middle-aged and older adults. Serum lipopolysaccharide (LPS), diamine oxidase (DAO) and D-lactate were detected to evaluate intestinal permeability. Gut microbiota (GM) and its potential interaction were also analyzed in the decision tree model. HGS was negatively correlated with DAO (r = -0.396, p < 0.01) in males. The negative association between HGS and DAO remained significant with the adjustment of covariates (β = -1.401, p < 0.05). Serum DAO and LPS were both negatively associated with HGS in middle-aged and older males, with the significant interactions of GM in the decision tree model, and D-lactate showed a negative association with HGS in females. Therefore, intestinal permeability was associated with the loss of skeletal muscle strength in middle-aged and older adults, and serum DAO may be a novel predictor for the loss of skeletal muscle strength in middle-aged and older males.