An in vitro reporter gene assay based on human breast cancer T47D cells (ER-CALUX®) was applied to examine the ability of diesel exhaust to induce or inhibit estrogen receptor (ER)-mediated gene expression. Exhaust from a heavy-duty diesel engine was either treated by iron- or copper/iron-catalyzed diesel particulate filters (DPFs) or studied as unfiltered exhaust. Collected samples included particle-bound and semivolatile constituents of diesel exhaust. Our findings show that all of the samples contained compounds that were able to induce ER-mediated gene expression as well as compounds that suppressed the activity of the endogenous hormone 17β-estradiol (E2). Estrogenic activity prevailed over antiestrogenic activity. We found an overall ER-mediated activity of 1.63 ± 0.31 ng E2 CALUX equivalents (E2-CEQs) per m3 of unfiltered exhaust. In filtered exhaust, we measured 0.74 ± 0.07 (iron-catalyzed DPF) and 0.55 ± 0.09 ng E2-CEQ m−3 (copper/iron-catalyzed DPF), corresponding to reductions in estrogenic activity of 55 and 66%, respectively. Our study demonstrates that both catalytic DPFs lowered the ER-mediated endocrine-disrupting potential of diesel exhaust.