While transfer-RNAs (tRNAs) are known to transport amino acids to ribosome, new functions are being unveiled from tRNAs and their fragments beyond protein synthesis. Here we show that phosphorylation of 90-kDa RPS6K (ribosomal proteins S6 kinase) was enhanced by tRNA^Leu overexpression under amino acids starvation condition. The phosphorylation of 90-kDa RPS6K was decreased by siRNA specific to tRNA^Leu and was independent to mTOR (mammalian target of rapamycin) signaling. Among the 90-kDa RPS6K family, RSK1 (ribosomal S6 kinase 1) and MSK2 (mitogen-and stress-activated protein kinase 2) were the major kinases phosphorylated by tRNA^Leu overexpression. Through SILAC (stable isotope labeling by/with amino acids in cell culture) and combined mass spectrometry analysis, we identified EBP1 (ErbB3-binding protein 1) as the tRNA^Leu-binding protein. We suspected that the overexpression of free tRNA^Leu would reinforce ErbB2/ErbB3 signaling pathway by disturbing the interaction between ErbB3 and EBP1, resulting in RSK1/MSK2 phosphorylation, improving cell proliferation and resistance to death. Analysis of samples from patients with breast cancer also indicated an association between tRNA^Leu overexpression and the ErbB2-positive population. Our results suggested a possible link between tRNA^Leu overexpression and RSK1/MSK2 activation and ErbB2/ErbB3 signaling.