We investigated the prevalence of Escherichia coli O157:H7 in free-ranging red deer in south-central Spain, to assess their potential as reservoir hosts of sorbitol-fermenting (SF) E. coli O157:H7 strains, which are emerging causes of hemolytic uremic syndrome in Europe. Fecal samples from 264 hunter-harvested Iberian red deer (Cervus elaphus) were collected in 25 different game estates and examined for E. coli O157:H7 by culture and PCR. E. coli O157:H7 was detected and isolated in 4 of the 25 game estates sampled (16%) and the isolates obtained (four in total) were further phenogenotypically characterized. One of them was biochemically typical of E. coli O157:H7, that is, neither fermented sorbitol nor exhibited β-glucuronidase (GUD) activity, and carried genes encoding Shiga toxins (Stx) 1 and 2, the intimin subtype γ1, the enterohemorrhagic E. coli (EHEC)-hemolysin, and the ter gene cluster. The rest of the isolates (three of four) fermented sorbitol, exhibited GUD activity after 18-24 h incubation, and carried genes encoding the intimin subtype γ1 and the EHEC-hemolysin, although no Stx-encoding genes were detected. All these atypical isolates carried the sfp gene cluster, lacked the ter gene cluster, and were unable to grow on cefixime tellurite sorbitol MacConkey agar, which are typical features of SF E. coli O157:H7 strains isolated from patients. In total, SF, GUD-positive, Stx-negative E. coli O157:H7 strains were isolated in 3 of the 25 game estates sampled (12%), with an overall sample-level prevalence of 1.1% (3/264). Our findings indicate that free-ranging red deer may be one of the possible reservoir hosts of Stx-negative derivatives of SF E. coli O157:H7.