Background:Based on the studies which have investigated conscious and unconscious processes, simple arithmetic operations such as addition and multiplication can be automatically processed in the brain and affect subsequent responses. However, most studies have focused on addition and multiplication of one-digit numbers. In this research we used subliminal priming paradigm to assess automatic retrieval of subtraction operation for the first time. Objectives: The aim of this study was to use a subliminal priming paradigm in a naming task and investigate the automatic and unconscious processing of the subtraction operation. Research of this kind can help us determine different levels of unconscious and conscious processing in the brain. Materials and Methods: Forty-five graduate student in psychology at the Faculty of Education and Psychology, University of Tabriz (between 18 and 25 years; mean 20.7, SD=2.7) participated in the experiment. For presenting the stimuli, an open-source software (DMDX) was used and presented on a 15-inch monitor. In the experiment, in the congruent condition, the prime was congruent with the target in terms of subtraction calculation result and in the incongruent condition there was no logical connection between the two stimuli. The vocal reaction time (RT) of participants was recorded and paired t-test analysis was conducted for comparison of the two conditions. Results: The data showed that naming the target by the participants is carried out faster when the two stimuli are congruent with each other in terms of the result of the operation. Conclusion: These findings may have implications on the levels of mathematical operations. In conclusion it seems that the calculation of one-digit numbers can happen at the level of simple neuronal circuits and may be carried out without conscious-awareness. The findings confirm the fact that calculating subtraction for one-digit numbers does not require conscious effort and can be processed automatically.