Arjunolic acid (AA) is a pentacyclic triterpenoid with promising anticancer properties. A series of novel AA derivatives containing a pentameric A-ring with an enal moiety, combined with additional modifications at C-28, were designed and prepared. The biological activity on the viability of human cancer and non-tumor cell lines was evaluated in order to identify the most promising derivatives. Additionally, a preliminary study of the structure-activity relationship was carried out. The most active derivative, derivative 26, also showed the best selectivity between malignant cells and non-malignant fibroblasts. For compound 26, the anticancer molecular mechanism of action in PANC-1 cells was further studied and the results showed that this derivative induced a cell-cycle arrest at G0/G1 phase and significantly inhibited the wound closure rate of PANC-1 cancer cells in a concentration-dependent manner. Additionally, compound 26 synergistically increased the cytotoxicity of Gemcitabine, especially at a concentration of 0.24 μM. Moreover, a preliminary pharmacological study indicated that at lower doses this compound did not demonstrate toxicity in vivo. Taken together, these findings suggest that compound 26 may be a valuable compound for the development of new pancreatic anticancer treatment, and further studies are needed to explore its full potential.