Nanosecond pulsed electric field (nsPEF) is a novel non‐thermal tumor ablation technique. However, how nsPEF affect cell physiology at different environmental temperature is still kept unknown. But this issue is of critical clinical practice relevance. This work aim to investigate how nsPEF treated cancer cells react to different environmental temperatures (0°C, 4°C, 25°C and 37°C). Their cell viability, apoptosis, mitochondrial membrane potential, and reactive oxygen species (ROS) were examined. Lower temperature resulted in higher apoptosis rate, decreased mitochondria membrane potential and increased ROS levels. Sucrose and N‐acetylcysteine (NAC) pre‐incubation inhibit ROS generation and increase cell survival, protecting nsPEF‐treated cells from low temperature‐caused cell death. This work provides an experimental basis for hypothermia and fluid transfusion during nsPEF ablation with anesthesia. This article is protected by copyright. All rights reserved