Background: TMPRSS2 plays an important role in the viral entry mechanisms of influenza viruses and coronaviruses. Therefore, TMPRSS2 seems to be a suitable antiviral drug target. To exclude possible side effects of TMPRSS2 truncation in an early stage of drug in-vitro testing, this study aims to analyze the impact of TMPRSS2 truncation via antisense peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) targeting immune cells, using the example of regulatory T cells (Treg). Methods: TMPRSS2 was truncated in human Tregs using a splice-modulating PPMO. Effects on Treg function were analyzed by evaluation of surface marker and transcription factor expression, cytokine secretion, and effector cell suppression capability. Results: PPMO treatment led to a slight concentration-dependent toxicity in Tregs. Tregs with truncated TMPRSS2 behave similarly to untreated and control PPMO-treated cells in the analyzed assays. Conclusions: Treg function is not altered after TMPRSS2 truncation and therefore, no unwanted side effects in regard of Tregs are expected when using TMPRSS2-truncating PPMO as an anti-viral drug.