Targeted anticancer therapeutics offer the advantage of reducing cytotoxic side effects to normal cells by directing the cytotoxic payload selectively to cancer cells. Designed ankyrin repeat proteins (DARPins) are promising non‑immunoglobulin‑based scaffold proteins for payload delivery to cancer‑associated molecular targets. Epithelial cell adhesion molecule (EpCAM) is overexpressed in 40‑60% of prostate cancers (PCs) and is associated with metastasis, increased risk of PC recurrence and resistance to treatment. Here, we investigated the use of DARPin Ec1 for targeted delivery of Pseudomonas exotoxin A variant (LoPE) with low immunogenicity and low non‑specific toxicity to EpCAM‑expressing prostate cancer cells. Ec1‑LoPE fusion protein was radiolabeled with tricarbonyl technetium‑99m and its binding specificity, binding kinetics, cellular processing, internalization and cytotoxicity were evaluated in PC‑3 and DU145 cell lines. Ec1‑LoPE showed EpCAM‑specific binding to EpCAM‑expressing prostate cancer cells. Rapid internalization mediated potent cytotoxic effect with picomolar IC50 values in both studied cell lines. Taken together, these data support further evaluation of Ec1‑LoPE in a therapeutic setting in a prostate cancer model in vivo.