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Tick-borne Encephalitis (TBE) is a zoonotic disease caused by the Tick-borne Encephalitis virus (TBEV), which affects the central nervous system of both humans and animals. Currently, there is no specific therapy for patients with TBE, with symptomatic treatment being the primary approach. In this study, the effects of minocycline (MIN), which is a kind of tetracycline antibiotic, on TBEV propagation and cellular protection in TBEV-infected cell lines were evaluated. Indirect immunofluorescence, virus titers, and RT-qPCR results showed that 48 h post-treatment with MIN, TBEV replication was significantly inhibited in a dose-dependent manner. In addition, the inhibitory effect of MIN on different TBEV multiplicities of infection (MOIs) in Vero cells was studied. Furthermore, the transcriptomic analysis and RT-qPCR results indicate that after incubation with MIN, the levels of TBEV and <i>CALML4</i> were decreased, whereas the levels of calcium channel receptors, such as <i>RYR2</i> and <i>SNAP25</i>, were significantly increased. MIN also regulated MAPK-ERK-related factors, including <i>FGF2</i>, <i>PDGFRA</i>, <i>PLCB2</i>, and p-ERK, and inhibited inflammatory responses. These data indicate that administering MIN to TBEV-infected cells can reduce the TBEV level, regulate calcium signaling pathway-associated proteins, and inhibit the MAPK-ERK signaling pathway and inflammatory responses. This research offers innovative strategies for the advancement of anti-TBEV therapy.