There has been a significant increase in the prevalence of Enterobacteriaceae that produce CTX-M-type extended-spectrum β-lactamases. The objective of this study was to evaluate risk factors for infection or colonization with CTX-M-positive Escherichia coli. A case-control study was conducted within a university system from 1 January 2007 to 31 December 2008. All patients with clinical cultures with E. coli demonstrating resistance to extended-spectrum cephalosporins were included. Case patients were designated as those with cultures positive for CTX-M-positive E. coli, and control patients were designated as those with non-CTX-M-producing E. coli. Multivariable logistic regression analyses were performed to evaluate risk factors for CTX-M-positive isolates. A total of 83 (56.8%) of a total of 146 patients had cultures with CTX-M-positive E. coli. On multivariable analyses, there was a significant association between infection or colonization with CTX-M-type β-lactamase-positive E. coli and receipt of piperacillin-tazobactam in the 30 days prior to the culture date (odds ratio [OR], 7.36; 95% confidence interval [CI], 1.61 to 33.8; P = 0.01) and a urinary culture source (OR, 0.36; 95% CI, 0.17 to 0.77; P = 0.008). The rates of resistance to fluoroquinolones were significantly higher in isolates from case patients than in isolates from control patients (90.4 and 50.8%, respectively; P < 0.001). We found that nonurinary sources of clinical cultures and the recent use of piperacillin-tazobactam conferred an increased risk of colonization or infection with CTX-M-positive E. coli. Future studies will need to focus on outcomes associated with infections due to CTX-M-positive E. coli, as well as infection control strategies to limit the spread of these increasingly common organisms.