Acetaminophen (APAP) given during chronic exercise reduces skeletal muscle collagen and cross-linking in rats. We propose that the effect of APAP on muscle ECM may, in part, be mediated by dysregulation of the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). The purpose of this study was to evaluate the impact of APAP consumption during acute resistance exercise (RE) on several regulators of the ECM in human skeletal muscle. In a double-blinded, placebo-controlled, randomized cross-over design, recreationally active men (n=8, 25±2yr) performed two trials of knee extension. Placebo (PLA) or APAP (1000mg/6h) was given for 24 hours prior to and immediately following RE. Vastus lateralis biopsies were taken at baseline, 1-hr, and 3-hr post-RE. RT-qPCR was used to determine differences in mRNA expression. MMP-2, COL1A1, and COL3A1 mRNA expression were not altered by exercise or APAP (p>0.05). When compared to PLA, TIMP-1 expression was lower at 1-hr post-RE during APAP conditions but greater than PLA at 3-hr post-RE (p<0.05). MMP-9 expression and protein levels were elevated at 3-hr post-RE independent of treatment (p<0.05). LOX expression was greater at 3-hr post-RE during APAP consumption (p<0.05) when compared to PLA. MMP-2 and TIMP-1 protein were not altered by RE or APAP (p>0.05). Phosphorylation of ERK1/2 and p38-MAPK increased (p<0.05) with RE but were not influenced by APAP. Our findings do not support our hypothesis and suggest that short-term APAP consumption prior to RE has a small impact on the measured ECM molecules in human skeletal muscle following acute RE.