Background: Q fever is a generally neglected infection caused by Coxiella burnetii. Slaughterhouse workers exposed to livestock are among occupationally at-risk people. Objectives: This study was conducted to investigate the seroprevalence of anti-Coxiella burnetii (Q fever) IgG antibody among industrial slaughterhouse workers and factors affecting the risk of infection. Methods: In this cross-sectional study serum samples were taken from 91 individuals working at the central industrial abattoir in Mashhad, Iran using a convenient sampling method. Sera were kept at -80°C until assayed for specific anti-Coxiella burnetii IgG antibodies (phase 1) using the commercial ELISA kit. The participants filled out a checklist addressing potential risk factors of acquiring the infection. SPSS 11.5 was used for data analysis considering a significance level of P < 0.05. Results: The participants’ mean age was 38.7 ± 8 years. Fifty-six percent of the studied individuals (51 out of 91) were found positive for anti-Coxiella burnetii antibodies. The most prevalent cases were sheep (29, 57%) and cow (18, 35%) butchers. The odds of Q fever infection increased among those with a history of accidental hand cuts of more than five times during the previous years (OR = 2.56, CI95% = 1.02 - 6.33, P-value = 0.04) and those dealing with sheep as the primary livestock (OR = 2.9, CI95% = 1.09 - 7.66, P = 0.02). Conclusions: The high seropositivity rate of anti-Coxiella burnetii IgG reflects high exposure rate of workers to this potentially serious pathogen in slaughtherhouses; therefore, careful education, follow-up, and revision of decontamination policies and improved occupational care and environmental hygiene should be strictly implemented in slaughterhouses to reduce the risk.