Objective? To?investigate?the?antimicrobial?resistance?and?beta-lactamase?production?profile?in?the?gram-negative?bacilli?isolated from urinary tract infections in the Second People's Hospital of Gansu Province during the period from 2014 to 2015. The results will provided to clinicians for better antimicrobial treatment. Methods? The?bacterial?isolates?were?identified?via?conventional?laboratory?tests?or?automatic?identification?systems?and?subjected?to?antimicrobial?susceptibility?testing?by?using?Kirby-Bauer?method.?Three-dimensional test was used to detect the enzymes conferring antimicrobial resistance. The susceptibility testing results were interpreted according to the CLSI breakpoints issued in 2012. Results A total of 987 gram-negative strains were isolated from urinary tract infections, including E. coli (51.6%, 509/987), Enterobacter (11.0%, 109/987), P. aeruginosa (10.5%, 104/987), K. pneumoniae (9.9%, 98/987), P. mirabilis (9.3%, 92/987), C. freundii (4.7%, 46/987), and other gram-negative bacilli (2.9%, 29/987). ESBLs were produced?in?494?(50.1?%)?of?the?987?strains?of?gram?negative?bacilli.?Preliminary?screening?test?identified?243?AmpC?beta-lactamases?producing?strains,?and?135?(13.7?%)?strains?were?confirmed?by?three-dimensional?test.?Both?ESBLs?and?Amps?beta-lactamases?were?produced in 16 (1.6%) strains. The prevalence of ESBLs-producing strains was 79.6% in E. coli, 34.6% in P. aeruginosa and 37.8% in K. pneumoniae?isolates.?Metallo-β-lactamase?or?KPC?beta-lactamase?was?not?identified.?The?antimicrobial?resistance?was?serious?in?gram-negative bacilli, which showed relatively low resistance rate to imipenem (0.02%), amikacin (10.6%), cefoperazone-sulbactam (23.8 %), and nitrofurantoin (25.2 %). E. coli prevalence of levofloxacin,?ciprofloxacin?resistance?respectively?were?89.8%,?91.8%. The gram-negative bacilli from urinary tract also showed various levels of resistance to cephalosporins. Conclusions The gram-negative bacilli isolated from urinary tract infections are mainly E. coli. The gram-negative isolates show high level antimicrobial resistance and high prevalence of beta-lactamases. Imipenem, amikacin, cefoperazone-sulbactam, and nitrofurantoin still have very high antibacterial activity against these isolates in vitro.