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Abstract Background Needle stick injury (NSI) is the most common cause of infection with blood-borne pathogens (BBP) among healthcare workers (HCWs). This study aimed to assess the prevalence of NSI and it’s contributing factors among HCWs of hemodialysis (HD) units in southwest Iran. Methods A cross-sectional study was performed in 13 HD centers in Shiraz, Iran. A total of 122 employees were enrolled in our study. We used self-administrated questionnaires to collect data about demographics, experiences regarding NSIs, and general health status. The statistical tests used in this study were Chi-square and Independent T-test. A P-value 10 years (p = 0.040), and those who graduated earlier (p = 0.031). The intravenous injection was the most common procedure leading to NSI, and being in a hurry was the most common cause. The average general health was 3.7 ± 3.2, higher among those not exposed to NSI (p = 0.042). Conclusion NSI is a prevalent hazard in HCWs of HD units. The high rate of NSI and unreported cases, besides the lack of adequate information, indicates the necessity of implementing protocols and strategies for improving the safety of this personnel. It is difficult to compare the result of this study with those performed among HCWs in other settings; hence, further studies are needed to determine whether HCWs of these units are more exposed to NSIs.