Abstract: Objective Using network pharmacology and molecular docking techniques to predict the potential mechanism by which active components in Piper (Piper nigrum L., PN) regulate ferroptosis to alleviate depression. Methods Firstly, the chemical composition of Piper was obtained from the Traditional Chinese Medicine System Pharmacology Database (TCMSP), and disease-related genes were obtained using the Online Mendelian Inheritance Database for Humans (OMIM), Genecards, and FerrDB databases. The active ingredient-target-disease network was mapped using Cytoscape software, the protein interaction network (PPI) was mapped using the STRING database, and gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out using the open-source bioinformatics software Bioconductor. Next, molecular docking was conducted to validate the binding capacity between the core targets and their corresponding active components. Finally, we established a chronic restraint stress (CRS) mouse model, treated with different concentrations of PN (75, 150, and 300 mg/kg) for four weeks, followed by behavioral assessments and qPCR to verify the expression of core genes. Results We identified nine active components from PN, corresponding to 27 targets. There are 8,377 targets related to depression and 547 targets related to ferroptosis, resulting in an intersection of 25 target genes. KEGG enrichment analysis revealed that these core targets are primarily enriched in signaling pathways such as cholinergic synapses, serotonergic synapses, and neuroactive ligand-receptor interactions. Molecular docking results indicate that the main active components of PN exhibit strong binding affinity with the targets CHRM2, SLC6A4, PTGS2, and SLC6A2. Behavioral assessments demonstrated that PN significantly increased the saccharin preference index in treated mice, reduced immobility time in the tail suspension and forced swimming tests, and enhanced exploratory behavior in the open field test. Neurotransmitter analysis revealed that PN significantly elevated serotonin and acetylcholine levels in the hippocampus of mice. qPCR results showed that PN can downregulate the mRNA expression of SLC6A4 and PTGS2. Conclusion PN may regulate ferroptosis and improve depressive-like behavior in mice by modulating serotonin and acetylcholine levels, participating in immune regulation, and exerting neuroprotective effects.