Aim The acute myocellular responses of caffeine supplementation during resistance exercise (RE) have not been investigated. beta(2)-Adrenergic receptors (beta(2)AR) may be a target of the stimulatory effects of caffeine and stimulate bioenergetic pathways including protein kinase A (PKA), and mitogen-activated protein kinases (MAPK). Purpose Elucidate the effects of pre-workout supplementation on signaling responses to an acute RE bout. Methods In a randomized, counter-balanced, double-blind, placebo-controlled, within-subject crossover study, ten resistance-trained males (mean +/- SD; age = 22 +/- 2.4 years, height = 175 +/- 7 cm, body mass = 84.1 +/- 11.8 kg) consumed a caffeine containing multi-ingredient pre-workout supplement (SUPP) or color and flavor matched placebo (PL) 60 min prior to an acute RE bout of barbell back squats. Pre- and post-exercise muscle biopsies were analyzed for the phosphorylation (p-) of beta(2)AR, PKA, and MAPK (ERK, JNK, p38). Epinephrine was determined prior to supplementation (baseline; BL), after supplementation but prior to RE (PRE), and immediately after RE (POST). Results Epinephrine increased at PRE in SUPP (mean +/- SE: 323 +/- 34 vs 457 +/- 68 pmol/l; p = 0.028), and was greatest at POST in the SUPP condition compared to PL (5140 +/- 852 vs 2862 +/- 498 pmol/l; p = 0.006). p-beta(2)AR and p-MAPK increased post-exercise (p < 0.05) with no differences between conditions (p > 0.05). Pearson correlations indicated there was a relationship between epinephrine and p-beta(2)AR in PL (r = - 0.810; p = 0.008), and p-beta(2)AR and ERK in SUPP (r = 0.941; p < 0.001). Conclusion Consumption of a caffeine containing pre-workout supplement improves performance, possibly through increases in pre-exercise catecholamines. However, the acute myocellular signaling responses were largely similar post-exercise.