Colorectal cancer (CRC) is a common and intractable malignancy with a high mortality risk. Conventional chemotherapeutics are effective for patients with early stage CRC, but the majority of deaths of CRC patients are linked to acquired drug resistance or metastasis occurrence. Asperphenin B (1), a lipopeptidyl benzophenone isolated from a marine-derived Aspergillus sp. fungus, reportedly possesses antiproliferative activity against cancer cells. However, its antitumor activity and the underlying molecular mechanisms remain unexplored. In this study, 1 induced G2/M phase cell cycle arrest and subsequent apoptotic cell death and inhibited tumor growth in a xenograft model. The 1-induced G2/M phase arrest was associated with the regulation of checkpoint proteins, including Chk1/2 and Cdc25c. The 1-induced apoptosis was correlated with an upregulation of p53 and cleaved caspases and a downregulation of survivin. Further experiments revealed that 1-mediated suppression of migration and invasion of metastatic HCT116 cells was partially associated with the downregulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The antimetastatic potential of 1 was also confirmed by E-cadherin upregulation and N-cadherin and Snail downregulation, which were in turn associated with the GAPDH regulation. These findings highlight the potential use of 1 as a novel candidate for treating metastatic CRC with the modulation of GAPDH function.