BackgroundTestis is the only organ supporting sperm production and with the largest number of proteins and tissue-specific proteins in animals. In our previous studies, we have found that knockdown ofocnus(ocn), a testis-specific gene, resulted in much smaller testis with no germ cells inDrosophila melanogaster. However, the molecular consequences ofocnknockdown in fly testes are unknown.ResultsIn this study, through iTRAQ quantitative proteomics sequencing, 606 proteins were identified from fly abdomens as having a significant and at least a 1.5-fold change in expression afterocnknockdown in fly testes, of which 85 were up-regulated and 521 were down-regulated. Among the differential expressed proteins (DEPs), apart from those proteins involved in spermatogenesis, the others extensively affected biological processes of generation of precursor metabolites and energy, metabolic process, and mitochondrial transport. Protein-protein interaction (PPI) analyses of DEPs showed that several kinases and/or phosphatases interacted with Ocn. Re-analyses of the transcriptome revealed 150 differential expressed genes (DEGs) appeared in the DEPs, and their changing trends in expressions afterocnknockdown were consistent. Many common down-regulated DEGs and DEPs were testis-specific or highly expressed in the testis ofD. melanogaster.Quantitative RT-PCR (qRT-PCR) confirmed 12 genes appeared in both DEGs and DEPs were significantly down-regulated afterocnknockdown in fly testes. Furthermore, 153 differentially expressed phosphoproteins (DEPPs), including 72 up-regulated and 94 down-regulated phosphorylated proteins were also identified (13 phosphoproteins appeared in both up- and down-regulated groups due to having multiple phosphorylation sites). In addition to those DEPPs associated with spermatogenesis, the other DEPPs were enriched in actin filament-based process, protein folding, and mesoderm development. Some DEPs and DEPPs were involved in Notch, JAK/STAT, and cell death pathways.ConclusionsGiven the drastic effect of theocnknockdown on tissue development and testis cells composition, the differences in protein abundance in theocnknockdown flies might not necessarily be the direct result of differential gene regulation due to the inactivation ofocn. Nevertheless, our results suggest that the expression ofocnis essential forDrosophilatestis development and that its down-regulation disturbs key signaling pathways related to cell survival and differentiation. These DEPs and DEPPs identified may provide significant candidate set for future studies on the mechanism of male reproduction of animals, including humans.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09386-2.