Enhanced cellular glucose uptake is a frequent characteristic of malignant cells. The Akt substrate of 160 kDa (AS160) is a newly discovered substrate for the protein kinase AKT and phosphorylation of AS160 (p-AS160) was recently recognized to play an important role in glucose transport. However, studies on AS160 in cancer do not yet exist. The aim of the present study was to investigate the p-AS160 level and its correlation with clinicopathological parameters and various biological markers in breast cancer. Results showed that in breast cancer, phosphorylation of AS160 at the key residue T642 was significantly (p < 0.001) higher than that in normal adjacent tissues. P-AS160 staining was positive in 75 of 81 cases (92.6%), including 32 with weak-(score 1), 31 with moderate-(score 2) and 12 with strong immunoreactivity (score 3). P-AS160 was inversely correlated with patient age (p = 0.041) and positively correlated with tumor size (p = 0.013) and the cell proliferation marker Ki-67 (MIB-1) (p < 0.001). This is the first study of AS160 in cancer. Our results show that AS160 phosphorylation level is frequently increased in breast cancer. These results implicate a possible role of AS160 in human breast tumorigenesis and suggest that p-AS160 might be useful as a marker and a potential novel treatment target for breast cancer.