Aging and obesity contribute to insulin resistance with skeletal muscle being critically important for maintaining whole-body glucose homeostasis. Both exercise and weight loss are lifestyle interventions that can affect glucose metabolism. The purpose of this study was to examine the effects of a six-month trial of aerobic exercise training or weight loss on signaling pathways in skeletal muscle in the basal condition and during hyperinsulinemia during a glucose clamp in middle-aged and older adults. Overweight and obese men and women aged 50-70 years were randomly allocated and completed six months of either weight loss (WL) (n = 18) or 3x/week aerobic exercise training (AEX) (n = 17). WL resulted in 10% weight loss and AEX increased maximal oxygen consumption (VO2max) (both p < 0.001). Insulin sensitivity (hyperinsulinemic-euglycemic 80 mU·m-2·min-1 clamp) increased in WL and AEX (both p < 0.01). In vivo insulin stimulation increased phosphorylation/total protein ratio (P/T) of protein kinase B (Akt), glycogen synthase kinase 3 beta (GSK-ß3), 70 kDa ribosomal protein S6 kinase (p70S6k), insulin receptor substrate 1 (IRS-1), and insulin receptor (IR) expression (all p < 0.05) but not P/T extracellular regulated kinase ½ (ERK1/2), c-jun N-terminal kinases (JNK), p38 mitogen-activated protein kinases (p38), or insulin-like growth factor 1 receptor (IGF-1R). There were differences between WL and AEX in the change in basal Akt P/T (p = 0.05), GSK-3ß P/T ratio (p < 0.01), p70S6k (p < 0.001), ERK1/2 (p = 0.01) P/T ratio but not p38, JNK, IRS-1, and IGF-1R P/T ratios. There was a difference between WL and AEX in the insulin stimulation changes in GSK3 which increased more after WL than AEX (p < 0.05). In the total group, changes in M were associated with changes in basal total GSK-3ß and basal total p70Sk as well as insulin stimulation of total p70Sk. Protein signaling in skeletal muscle provides insight as to mechanisms for improvements in insulin sensitivity in aging and obesity.