Our aim was to examine the effect of betaine supplementation on selected circulating hormonal measures and Akt muscle signaling proteins after an acute exercise session. Twelve trained men (age 19.7 ± 1.23 years) underwent 2 weeks of supplementation with either betaine (B) (1.25 g BID) or placebo (P). Following a 2-week washout period, subjects underwent supplementation with the other treatment (B or P). Before and after each 2-week period, subjects performed an acute exercise session (AES). Circulating GH, IGF-1, cortisol, and insulin were measured. Vastus lateralis samples were analyzed for signaling proteins (Akt, p70 S6k, AMPK). B (vs. P) supplementation approached a significant increase in GH (mean ± SD (Area under the curve, AUC), B: 40.72 ± 6.14, P: 38.28 ± 5.54, p = 0.060) and significantly increased IGF-1 (mean ± SD (AUC), B: 106.19 ± 13.45, P: 95.10 ± 14.23, p = 0.010), but significantly decreased cortisol (mean ± SD (AUC), B: 1,079.18 ± 110.02, P: 1,228.53 ± 130.32, p = 0.007). There was no difference in insulin (AUC). B increased resting Total muscle Akt (p = 0.003). B potentiated phosphorylation (relative to P) of Akt (Ser(473)) and p70 S6 k (Thr(389)) (p = 0.016 and p = 0.005, respectively). Phosphorylation of AMPK (Thr(172)) decreased during both treatments (both p = 0.001). Betaine (vs. placebo) supplementation enhanced both the anabolic endocrine profile and the corresponding anabolic signaling environment, suggesting increased protein synthesis.