Background: Ventilator-associated pneumonia (VAP) is a common nosocomial infection among hospitalized patients who have undergone intubation and mechanical ventilation for more than 48 hours. Patients admitted to the intensive care unit (ICU) are at risk of developing life-threatening VAP due to specific conditions, especially with Gram-negative pathogens with advanced drug resistance. Hereby, the control of these agents and its monitoring is of particular importance. In this study, the pattern of antibiotic resistance of Gram-negative bacteria isolated from tracheal culture of patients with VAP investigated in ICU of Imam Khomeini Hospital of Ahwaz. Materials and Methods: In this cross-sectional study, tracheal samples were collected during April 2016 to April 2017 from patients who were on mechanical ventilation in ICU of Imam Khomeini Hospital in Ahwaz, Khuzestan province, southwest of Iran. After isolation, bacterial strains were identified using biochemical tests. Then, antimicrobial resistance pattern of these isolates investigated using standard disc diffusion according to clinical and laboratory standards institute 2016 (CLSI 2016) guidelines. Results: A total of 111 bacterial isolates were identified which were as following; Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, STENOTROPHOMONAS maltophilia, and Serratia marcescens, with prevalence of 54%, 19. 8%, 14. 4%, 6. 4%, 4. 5%, and 0. 9%, respectively. Antibiotic susceptibility test of isolates showed that almost all isolates had high resistance to treatment antibiotics and were multi-drug resistance (MDR). The A. baumannii isolates were resistant to ciprofloxacin and piperacillin-tazobactam, but ampicillin-tazobactam had a good effect. Conclusion: The results of this study showed that patients admitted to ICU due to their conditions of treatment are more likely to develop VAP by Gram-negative pathogens. The empirical treatment of VAP due to predominant bacterial causes and emerging drug resistance has become more challenging. It requires to use of multidrug regimens for routine clinical practice. It should be noted that in order to appropriate antimicrobial therapy, precise and correct diagnosis is very important.