The antitumour effect of melittin (MEL) has recently attracted considerable attention. Nonetheless, information regarding the functional role of MEL in bladder cancer (BC) is currently limited. Herein, we investigated the effect of MEL on critical module genes identified in BC. In total, 2015 and 4679 differentially expressed genes (DEGs) associated with BC were identified from the GSE31189 set and The Cancer Genome Atlas database, respectively. GSE-identified DEGs were mapped and analysed using Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes analyses to determine BC-involved crucial genes and signal pathways. Coupled with protein-protein interaction network and Molecular Complex Detection analyses, Modules 2 and 4 were highlighted in the progression of BC. In in-vitro experiments, MEL inhibited the proliferation, migration, and invasion of UM-UC-3 and 5637 cells. The expression of NRAS, PAK2, EGFR and PAK1 in Module 4-enriched in the MAPK signalling pathway-was significantly reduced after treatment with MEL at concentrations of 4 or 6 μg/mL. Finally, quantitative reverse transcription-polymerase chain reaction and Western blotting analyses revealed MEL inhibited the expression of genes at the mRNA (ERK1/2, ERK5, JNK and MEK5), protein (ERK5, MEK5, JNK and ERK1/2) and phosphorylation (p-ERK1/2, p-JNK, and p-38) levels. This novel evidence indicates MEL exerts effects on the ERK5-MAK pathway-a branch of MAPK signalling pathway. Collectively, these findings provide a theoretical basis for MEL application in BC treatment.