Human serum albumin (HSA) fusion protein is a viable and effective approach to target and inhibit essential intracellular pathways. It has previously been shown that an HSA fusion protein containing a p53-reactivating peptide (rHSA-p53i) retains the binding activity to MDM2 and MDMX, resulting in p53 transcription-dependent apoptosis. Here, we demonstrate that rHSA-p53i is able to bind and neutralize anti-apoptotic Bcl-2 family proteins, Bcl-xL and Mcl-1. This interaction displaces pro-apoptotic Bak and subsequently leads to intrinsic apoptosis via mimicking a p53 transcription-independent pathway. Cytotoxicity induced by rHSA-p53i, via p53 transcription dependent and independent apoptotic pathways, is irrespective of the p53 status in MDA-MB-231, HeLa, and SJSA-1 cells possessing either mutant, deficient, or wild-type p53. The therapeutic potential is also confirmed by treating SJSA-1 and MDA-MB-231 xenograft mouse tumors with rHSA-p53i. These data reveal that rHSA-p53i interferes with at least four intracellular targets, making it a viable therapeutic protein for the treatment of a variety of cancers, as well as a carrier to deliver fatty acid-modified chemotherapeutics.