Mosquitoes host a wide diversity of insect-specific viruses alongside medically important arboviruses. While insect-specific viruses have been proposed as potential modulators of arbovirus transmission, the mechanisms underlying these interactions remain poorly understood, particularly in the context of persistent infections (longterm infection without clearance) that characterise many mosquito–virus associations. Here, we investigated interactions between insect-specific alphaviruses and arboviruses in mosquito cell lines. Insect-specific alphaviruses reduced the replication of related arboviruses only when they were persistently infecting cells. No strong interference was detected in sequential or simultaneous co-infection settings. To explore potential underlying mechanisms, we compared acute and persistent insect-specific virus infections. Differences were observed at multiple levels, including changes in host transcriptomic profiles and microRNA expression. In addition, small RNA sequencing revealed antiviral RNAi responses targeting ISVs, with differences between acute and persistent conditions. Overall, our results indicate differences in host responses associated with persistent insect-specific virus infections, which may influence subsequent arbovirus infections. Further work will be needed to determine how these responses contribute to interference phenotypes and whether they can be leveraged to understand better or modulate arbovirus transmission.