The Plasmodium proteasome represents a potential antimalarial drug target for compounds with activity against multiple life cycle stages. We screened a library of human proteasome inhibitors (peptidyl boronic acids) and compared activities against purified P. falciparum and human 20S proteasomes. We chose four hits that potently inhibit parasite growth and show a range of selectivities for inhibition of the growth of P. falciparum compared with human cell lines. P. falciparum was selected for resistance in vitro to the clinically used proteasome inhibitor, bortezomib, and whole genome sequencing was applied to identify mutations in the proteasome β5 subunit. Active site profiling revealed inhibitor features that enable retention of potent activity against the bortezomib-resistant line. Substrate profiling reveals P. falciparum 20S proteasome active site preferences that will inform attempts to design more selective inhibitors. This work provides a starting point for the identification of antimalarial drug leads that selectively target the P. falciparum proteasome.