ABSTRACT Electrochemotherapy (ECT) as a tumor treatment modality is approved for cutaneous and subcutaneous tumors. The purpose of the present study was to examine the effect of 900 MHz radiofrequency (RF) pulse-modulated by 217 Hz EMFs similar to those emitted by mobile phones on the mechanisms of ECT in vivo including: tumor hypoxia and immune system response, and on tumor volume.4 T1 cells were injected subcutaneously into the right flank of Balb/c mice. The mice were exposed to RF fields at specific absorption rate (SAR) 2 W/kg for 10 min/day and then treated with ECT. Two protocols of ECT were used: ((70 V/cm-5 kHz) and 70 V/cm-4 kHz)). Tumor hypoxia was analyzed through HIF-1α immuonohistochemistry assay. Interleukin 4 (IL-4) and IFN-γ levels were estimated by enzyme-linked immunosorbent assay (ELISA) technique to evaluate immune system response. Also, tumors volume changes were measured for 24 days following the treatment. The results showed that pulse-modulated RF fields could increase hypoxia induced by ECT, significantly (about 13% in ECT (70 V/cm-5 kHz) and 11% in ECT (70 V/cm-4 kHz)). However, these fields did not have significant effect on immune system response (the levels of IL-4 and IFN-γ) and tumor volume changes induced by ECT. Our results indicated that pulse-modulated RF fields could not affect tumor volume changes in ECT with the frequency of 5 kHz and voltage of 70 V/cm efficacy in vivo. However, investigating the role of other environmental intervening factors on this protocol of ECT is recommended in further studies.