There are substantial shortfalls in nurse staffing in US neonatal intensive care units (NICUs) relative to national guidelines. These are associated with higher rates of nosocomial infections among infants with very low birth weights. To study the adequacy of NICU nurse staffing in the United States using national guidelines and analyze its association with infant outcomes. Retrospective cohort study. Data for 2008 were collected by web survey of staff nurses. Data for 2009 were collected for 4 shifts in 4 calendar quarters (3 in 2009 and 1 in 2010). Sixty-seven US NICUs from the Vermont Oxford Network, a national voluntary network of hospital NICUs. All inborn very low-birth-weight (VLBW) infants, with a NICU stay of at least 3 days, discharged from the NICUs in 2008 (n = 5771) and 2009 (n = 5630). All staff-registered nurses with infant assignments. We measured nurse understaffing relative to acuity-based guidelines using 2008 survey data (4046 nurses and 10 394 infant assignments) and data for 4 complete shifts (3645 nurses and 8804 infant assignments) in 2009-2010. An infection in blood or cerebrospinal fluid culture occurring more than 3 days after birth among VLBW inborn infants. The hypothesis was formulated prior to data collection. Hospitals understaffed 31% of their NICU infants and 68% of high-acuity infants relative to guidelines. To meet minimum staffing guidelines on average would require an additional 0.11 of a nurse per infant overall and 0.34 of a nurse per high-acuity infant. Very low-birth-weight infant infection rates were 16.4% in 2008 and 13.9% in 2009. A 1 standard deviation-higher understaffing level (SD, 0.11 in 2008 and 0.08 in 2009) was associated with adjusted odds ratios of 1.39 (95% CI, 1.19-1.62; P < .001) in 2008 and 1.40 (95% CI, 1.19-1.65; P < .001) in 2009. Substantial NICU nurse understaffing relative to national guidelines is widespread. Understaffing is associated with an increased risk for VLBW nosocomial infection. Hospital administrators and NICU managers should assess their staffing decisions to devote needed nursing care to critically ill infants.