Though widely consumed, current research on the neural mechanisms of arecoline, caffeine, and nicotine remains limited, and the similarities and differences of these substances on the nervous system are still not clear. This study used RNA-seq to analyze the gene expression in the nucleus accumbens (NAc) of mice, and compared the behavioral changes through open field and conditioned place preference (CPP), exploring the effects of different psychoactive substances at transcriptional and behavioral levels. Gene Ontology enrichment analysis revealed that nicotine and caffeine significantly alter biological processes related to synaptic function, and KEGG pathway analysis showed that the differentially expressed genes in the nicotine-treated group were significantly more enriched in pathways related to substance dependence, with arecoline showing the least enrichment. Furthermore, only acute caffeine treatment significantly increased mouse activity, and only nicotine induced CPP. These results provided a scientific basis for evaluating arecoline, caffeine, and nicotine on the nervous system.