Urinary tract infections are common. Few published studies have demonstrated the change in Escherichia coli urinary isolate antimicrobial susceptibility over time within a given area and (or) population. The purpose of this study was to evaluate the change in susceptibility of E. coli clinical isolates obtained from urine specimens at a single institution over a period of 10 years. The microbiology laboratory information system at St. Boniface Hospital (Winnipeg, Manitoba, Canada) was searched retrospectively from 1 January 2000 to 31 December 2009, for all E. coli isolates from either a midstream or catheter urine source that had susceptibility testing performed. Only one isolate per patient was included during the entire study period. Antimicrobial susceptibility testing was carried out with either a Microscan instrument (pre-April 2004) or a Vitek instrument (May 2004 onwards). In total, 7353 E. coli urinary isolates were included for evaluation. Ciprofloxacin susceptibility declined significantly, from 99% in 2000 to 85% in 2009 (p < 0.0001). A small but statistically significant decline in susceptibility was also observed for ampicillin, cefazolin, trimethoprim-sulfamethoxazole, gentamicin, and nitrofurantoin. These data suggest that certain antimicrobials recommended for the treatment of urinary tract infections (ciprofloxacin, trimethoprim-sulfamethoxazole) may no longer be optimal.