Background: Glyphosate is the most widely used herbicide worldwide, both in domestic and industrial settings. Experimental research in animal models has demonstrated changes in muscle physiology and reduced contractile strength associated with glyphosate exposure, while epidemiological studies have shown associations between glyphosate exposure and adverse health outcomes in critical biological systems affecting muscle function.Methods: This study used data from a nationally representative survey of the non-institutionalized U.S. general population (NHANES, n = 2132). Urine glyphosate concentrations were determined by ion chromatography with tandem mass spectrometry. Hand grip strength (HGS) was measured using a Takei Dynamometer, and relative strength estimated as the ratio between HGS in the dominant hand and the appendicular lean mass (ALM) to body mass index (ALMBMI) ratio. Low HGS and low relative HGS were defined as 1 sex-, age-and race-specific SD below the mean. Physical function limitations were identified as significant difficulty or incapacity in various activities.Results: In fully-adjusted models, the Mean Differences (MD) and 95% confidence intervals [95%CI] per doubling increase in glyphosate concentrations were − 0.55 [-1.09, − 0.01] kg for HGS in the dominant hand, and − 0.90 [-1.58. − 0.21] kg for HGS/ALMBMI. The Odds Ratios (OR) [95% CI] for low HGS, low relative HGS and functional limitations by glyphosate concentrations were 1.27 [1.03, 1.57] for low HGS; 1.43 [1.05; 1.94] for low relative HGS; 1. 33 [1.08, 1.63] for stooping, crouching or kneeling difficulty; 1.17 [0.91, 1.50] for lifting or carrying items weighting up to 10 pounds difficulty; 1. 21 [1.01, 1.40] for standing up from armless chair difficulty; and 1.47 [1.05, 2.29] for ascending ten steps without pause difficulty. Conclusions: Glyphosate exposure may be a risk factor for decreased grip strength and increased physical functional limitations. More studies investigating the influence of this and other environmental pollutants on functional aging are needed.