Background With its complex and prevalent symptoms, chronic neuropathic pain (CNP) is frequently combined with negative emotions. Electroacupuncture (EA) is a widely-used and effective treatment for CNP and pain-induced comorbid anxiety-like behaviors (PABs). Although different effects may result from EA with different intensities, the underlying mechanisms remain unclear. Objective The study aimed to investigate the effects of EA with different intensities on CNP and PABs and the underlying mechanisms, by exploring the different genes through RNASeq in the spinal cord dorsal horn (SCDH) and periaqueductal gray (PAG). Methods A spared nerve injury (SNI) model was established using male C57BL/6 J mice, and treated with EA of different intensities (0.1 mA or 0.3 mA, both at 100 Hz). The von-Frey tests, open field tests and elevated plus maze tests were used to measure the mechanical paw withdrawal threshold (PWT) and PABs of the mice. The tissues of SCDH and PAG regions were extracted to identify the changes in gene expression, and biofunctional analysis of differentially expressed genes (DEGs) was further performed by RNA-seq analysis. The expression of key DEGs and corresponding proteins was assessed using quantitative real-time PCR (qPCR) and protein immunofluorescence detection, respectively. Results Both 0.1 mA and 0.3 mA EA at 100 Hz alleviated PABs compared with the SNI group. Furthermore, 0.3 mA EA was effective in increasing PWT in SNI mice, whereas 0.1 mA EA was not. A total of 108 DEGs was identified in the SCDH, and 254 DEGs were identified in the PAG. Specifically, the expression of Tnr in SNI mice decreased after the treatment with 0.3 mA EA at 100 Hz, and the expression of Ptprb, Net1, Abcb1a and Adgrf5 in SNI mice decreased in the PAG after the treatment with 0.1 mA EA at 100 Hz. Conclusion The effect of 0.3 mA EA on chronic pain may be related to the decrease of Tnr expression in SCDH, and the effect of 0.1 mA EA on PABs may be achieved by decreased expression of Ptprb and Net1 in PAG.