The aim of this study was to investigate the effects of long work hours and poor sleep characteristics on workplace injury. A total of 1891 male employees, aged 18–79 years (mean 45 years), in 296 small- and medium-scale businesses in a suburb of Tokyo were surveyed by means of a self-administered questionnaire during August–December 2002. Work hours and sleep characteristics, including daily sleep hours, subjective sleep sufficiency, sleep quality and easiness to wake up in the morning, were evaluated. Information on workplace injury in the past 1-year period was self-reported. The risk of workplace injury associated with work hours and poor sleep was estimated using multivariate logistic regression with odds ratio (ORs) and 95% confidence intervals as measures of associations. Compared with those working 6–8 h day−1 with good sleep characteristics, positive interactive effects for workplace injury were found between long work hours (>8–10 h day−1 or >10 h day−1) and short sleep duration (<6 h) [adjusted OR (aOR), 1.27–1.54], subjective insufficient sleep (aOR, 1.94–1.99), sleep poorly at night (aOR, 2.23–2.49) and difficulty waking up in the morning (aOR, 1.56–1.59). Long work hours (aOR, 1.31–1.48), subjective insufficient sleep (aOR, 1.49) and sleeping poorly at night (aOR, 1.72) were also independently associated with workplace injury. This study suggests that long work hours coupled with poor sleep characteristics are synergistically associated with increased risk of workplace injury. Greater attention should be paid to manage/treat poor sleep and reduce excessive work hours to improve safety at the workplace.