Tigecycline is one of the few remaining therapeutic options for extensively drug-resistant (XDR) Gram-negative bacilli (GNB). MICs of tigecycline to Acinetobacter baumannii have been reported to be elevated when determined by the Etest compared to determinations by the broth microdilution (BMD) method. The study aim was to compare the susceptibility of GNB to tigecycline by four different testing methods. GNB were collected from six health care systems (25 hospitals) in southeast Michigan from January 2010 to September 2011. Tigecycline MICs among A. baumannii, carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, and susceptible Enterobacteriaceae isolates were determined by Etest, BMD, Vitek-2, and MicroScan. Nonsusceptibility was categorized as a tigecycline MIC of ≥4 μg/ml for both A. baumannii and Enterobacteriaceae. The study included 4,427 isolates: 2,065 ESBL-producing Enterobacteriaceae, 1,105 A. baumannii, 888 susceptible Enterobacteriaceae, and 369 CRE isolates. Tigecycline nonsusceptibility among A. baumannii isolates was significantly more common as determined by Etest compared to that determined by BMD (odds ratio [OR], 10.3; P < 0.001), MicroScan (OR, 12.4; P < 0.001), or Vitek-2 (OR, 9.4; P < 0.001). These differences were not evident with the other pathogens. Tigecycline MICs varied greatly according to the in vitro testing methods among A. baumannii isolates. Etest should probably not be used by laboratories for tigecycline MIC testing of A. baumannii isolates, since MICs are significantly elevated with Etest compared to those determined by the three other methods.