Background <p>The role of melatonin (MLT) in modulating malignant phenotypes of carboplatin (CBP)-resistant ovarian cancer (OC) cells remains unexplored. This study therefore aimed to investigate effects of MLT on malignant biological behaviors in CBP-resistant OC cells and to preliminarily delineate underlying molecular mechanisms.</p> Methods <p>CBP-resistant A2780 and SKOV3 cell lines were first established, and their resistance index (RI) was determined by CCK-8 assay. Subsequently, effects of MLT were systematically evaluated: cell proliferation was assessed by CCK-8 assay; cell cycle distribution and apoptosis were analyzed by flow cytometry; and cell invasion and migration capabilities were examined using Transwell chambers. The potential mechanism by which MLT modulates malignant biological behaviors in CBP-resistant OC cells was elucidated through mRNA sequencing and bioinformatics analyses.</p> Results <p>CBP-resistant OC cell lines were successfully established. We demonstrated that MLT effectively suppressed key malignant phenotypes of CBP-resistant OC cells, including proliferation, cell cycle progression, invasion, and migration, while promoting apoptosis (<i>P</i> &lt; 0.05). Transcriptomic analyses demonstrated that MLT upregulated 548 genes and downregulated 119 genes in A2780/CBP cells, and upregulated 70 genes and downregulated 232 genes in SKOV3/CBP cells. Notably, a common set of 26 differentially expressed genes (DEGs) was consistently identified in both resistant cell lines. These shared DEGs were significantly enriched in the GO term “negative regulation of canonical Wnt signaling pathway” and KEGG pathway “amphetamine addiction”.</p> Conclusions <p>MLT inhibited malignant biological behaviors of CBP-resistant OC cells. The underlying mechanism likely involved the “negative regulation of canonical Wnt signaling pathway” (GO term) and “amphetamine addiction” (KEGG pathway).</p>