Gemcitabine has limited clinical benefits for pancreatic cancer patients. The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is important in cell proliferation and survival, and is frequently dysregulated in pancreatic cancer. To obtain insights into novel therapeutic strategies for treating pancreatic cancer, we investigated whether HS-104, a novel PI3K inhibitor, in combination with gemcitabine would show a synergistic effect in pancreatic cancer. We first evaluated the effect of gemcitabine alone or in combination with HS-104 on cell viability. When administered together, the two drugs synergistically inhibited the viability of AsPC-1 and PANC-1 cells, and decreased mitochondrial membrane potential, thereby inducing apoptosis. Compared to the treatment with either drug alone, the combination treatment resulted in apoptosis accompanied by increased levels of cleaved caspase-3 and Bax. These results were consistent with decreased expression of p-Akt, p-mTOR, p-Mek and p-Erk. Moreover, the combination treatment inhibited blood vessel formation in a Matrigel plug assay in mice. Furthermore, in vivo, the combination significantly inhibited tumor growth and enhanced apoptosis by increasing the number of TUNEL-positive cells, and cleaved caspase-3 together with decreasing the expression of angiogenesis- and proliferation-related effectors such as CD34 and PCNA in tumor tissues, compared with each drug alone. Taken together, our study demonstrates that the combination of gemcitabine and HS-104 had a synergistic anticancer effect and inhibited the PI3K/Akt and RAF/Mek pathways on pancreatic cancers. On the basis of our results, we suggest that the combination of these two drugs may be considered as a new therapeutic regimen for treating pancreatic cancer.