Ampicillin-sulbactam is commonly used as an empirical therapy for invasive infections where Escherichia coli is a potential pathogen. We evaluated the clinical and microbiologic characteristics of bloodstream infection due to E. coli, with focus on cases that were nonsusceptible to ampicillin-sulbactam and not producing extended-spectrum β-lactamase (ESBL). Of a total of 357 unique bacteremic cases identified between 2005 and 2008, 111 (31.1%) were intermediate or resistant to ampicillin-sulbactam by disk testing. In multivariate analysis, a history of liver disease, organ transplant, peptic ulcer disease, and prior use of ampicillin-sulbactam were independent risk factors for bloodstream infection with ampicillin-sulbactam-nonsusceptible E. coli. Among cases that received ampicillin-sulbactam as an empirical therapy, an early clinical response was observed in 65% (22/34) of susceptible cases but in only 20% (1/5) of nonsusceptible cases. Among 50 ampicillin-sulbactam-resistant isolates examined, there was no clonal relatedness and no evidence of production of inhibitor-resistant TEM (IRT). Instead, the resistance was attributed to hyperproduction of TEM-1 β-lactamase in the majority of isolates. However, promoter sequences of bla(TEM-1) did not predict resistance to ampicillin-sulbactam. While the plasmid copy number did not differ between representative resistant and susceptible isolates, the relative expression of bla(TEM-1) was significantly higher in two of three resistant isolates than in three susceptible isolates. These results suggest high-level bla(TEM-1) expression as the predominant cause of ampicillin-sulbactam resistance and also the presence of yet-unidentified factors promoting overexpression of bla(TEM-1) in these isolates.