In this work, isothermal aging of Sn-2.5Bi-1.4In-1Zn-0.3Ag (in wt% hereafter) solder with Cu substrate for 0, 0.5, 2, 10, 100 and 200 h were conducted, and the variation of intermetallic compounds (IMCs) and microhardness were investigated. For the as-soldered sample, the Cu6Sn5 and Cu5Zn8 layers formed at the interface and Cu6Sn5, Cu5Zn8, Ag–Sn–In, Ag–Zn–In IMCs and Bi particles were observed in the solder matrix. During aging treatment, the Cu5Zn8 layer decomposed and finally disappeared at the interface, and a new Cu3Sn layer appeared just below the Cu6Sn5 layer. Besides, in the solder matrix phase transformation from Cu5Zn8 into CuZn proceeded and the content of In in the Ag–Sn–In and Ag–Zn–In IMCs increased and Bi particles dispersed uniformly with the increasing aging time. Furthermore, the microhardness of the solder changed during aging owing to the observed phase evolution. These evolutions are always considered harmful to the reliability of the solder joints, especially the appearance and the growth of the Cu3Sn layer.