Aim To evaluate the effect of intracanal cryotherapy with negative pressure irrigation (EndoVac) on postendodontic pain after vital single-visit root canal treatment (RCT). Materials and methods A total of 75 single-rooted teeth with single root canal were treated endodontically. After root canal preparation with Protaper Universal rotary system and irrigation, teeth were divided randomly into three groups (n = 25) according to additional irrigation protocol as follows: Group I: No additional irrigation was applied (control); group II: A 20 mL of room temperature saline was irrigated during 5 minutes using EndoVac, and group III: A 20 mL of 2 to 4°C cold saline was irrigated during 5 minutes using EndoVac. Pain levels were assessed by visual analog scale (VAS) and verbal evaluation of pain questionnaire after 6, 12, 24, 48 hours, and 7 days of canal obturation. The data were then analyzed using Statistical Package for the Social Sciences (SPSS) 13.0 using Kruskal–Wallis and Mann–Whitney U tests at p-value of 0.05. Results The results showed that pain levels were high in groups I and II after 6 hours that decreased with time to almost diminish after 1 week, and on the other hand, group III showed no pain among different monitoring periods. Also pain levels in groups II were lower compared with group I after only 6 hours, with significance p < 0.05. Conclusion Postendodontic pain presented with highest values after 6 hours of treatment and reduced to almost nil after 1 week. Intracanal cryotherapy eliminated postendodontic pain clinically. Negative pressure reduced postendodontic pain after 6 hours of treatment. Clinical significance The outcome of this study indicates that the use of intracanal cryotherapy technique with negative pressure irrigation eliminates postendodontic pain after single-visit RCTs. How to cite this article Al-Nahlawi T, Abo Hatab T, Abd Alrazak M, Al-Abdullah A. Effect of Intracanal Cryotherapy and Negative Irrigation Technique on Postendodontic Pain. J Contemp Dent Pract 2016;17(12):990-996.