Abstract: Objectives To discuss the clinical characteristic, cause and measures to prevention and control of nosocomial infection in a neonatal intensive care unit (NICU). Methods Retrospectively analyzed an nosocomial infection outbreak of Klebsiella pneumoniae in NICU. Results From Sept. 3, 2010 to Oct. 3, 2010, there were 7 cases of hospital infection in 12 cases of sputum cultured Klebsiella Pneumoniae. The gestational age (GA) of 7 hospital infection cases was 28.5±2.6 week. The irth weight of infection cases was 941.4±309.8 g. The onset of infection was at 31.7±12.8 d of hospitalization. The nosocomial incidence was 2.41% in the hospital, which was 5.79% in preterm infants, 50.00% in GA <28w infants, and 42.86% in extremely low birth weight infant (ELBW). All sputum culture results were displayed as multi-drug resistant of Klebsiella pneumoniae, penicillin and third-generation cephalosporin antibiotic resistance rate of 75% to 100%. The resistance rates to penicillin and cephem antibiotics were 75% -100%, carbapenems was 58.3%, piperacillin/tazobactam was 25.0%. All nosocomial patients were cured. Conclusions GA <28w and ELBW infants are at increased risk of nosocomial infection in NICU. The emergence of carbapenems resistant Klebsiella Pneumoniae has been increasing with the widespread use of carbapenems. Hospital infection can be controlled by standardized medical behavior, which can decline the nosocomial infection incidence and mortality of preterm infants in NICU.