Patients feel uncomfortable with cervical manipulation, uterine distension and stimulation of peritoneum during hysterosalpingography (HSG) and experience lower abdominal pain during and after the procedure. Pain during the procedure has a negative effect on the adaptation of patients to treatment and physicians are trying to overcome this unpleasant situation. Therefore, the aim of this study was to compare the effect of midazolam and dexmedetomidine on reducing pain and spasm of fallopian tubes during and after HSG procedure in women with infertility. In a double-blind randomized controlled trial, 102 patients were randomly divided into two groups, midazolam and dexmedetomidine. The pain was recorded during injection and immediately after injection and 30 minutes after HSG, and then the complications of injection were recorded. Finally, the data were analyzed using SPSS version 20. Based on the results presented herein, no significant difference was found between the two groups in terms of vasovagal reaction, spasticity of the tube and the side of the spastic tube and uterine cavity anomalies (p <0.05). However, the pain showed a significant difference between the two groups during the injection, immediately or at 30 minutes after the procedure (p = 0.0001). The pain in the midazolam group was less than that of dexmedetomidine. Furthermore, there was no significant difference between the two groups regarding spasticity (p <0.05). There is a benefit in terms of pain reduction with the use of dexmedetomidine when comparing with midazolam injection. However, dexmedetomidine does not cause side effects in patients and can be used to reduce pain during injection.