This study investigated the incidence and risk to staff groups for sustaining needlestick injuries (NSIs) in the National University Hospital (NUH), Singapore. A retrospective cohort review of incident NSI cases was undertaken to determine the injury rate, causation, and epidemiological profile of such injuries. Analysis of the risk of sustaining recurrent NSI by occupation and location was done using the Cox proportional hazards model. There were 244 NSI cases in 5957 employees in NUH in 2014, giving an incidence rate of 4·1/100 healthcare workers (HCWs) per year. The incidence rate was highest for doctors at 21·3, and 2·7 for nurses; 40·6% of injuries occurred in wards, and 32·8% in operating theatres. There were 27 cases of repeated NSI cases. The estimated cost due to NSIs in NUH ranged from US$ 109 800 to US$ 563 152 in 2014. We conclude that creating a workplace environment where top priority is given to prevention of NSIs in HCWs, is essential to address the high incidence of reported NSIs. The data collected will be of value to inform the design of prevention programmes to reduce further the risk of NSIs in HCWs.