Rhabdomyosarcoma is the most common soft tissue sarcoma in childhood and young adulthood. Conventional treatment consisting of surgery, chemotherapy and radiotherapy can be insufficient, as long-term survival chances decrease dramatically when cancer recurrence occurs. Due to this fact, efficient treatment of this cancer is still a demanding issue, thus, novel and innovative therapies have to be considered as a part of combined treatment. In the present study, we present effective suicide gene therapy of rhabdomyosarcoma cell line Rh30 involving herpes simplex thymidine kinase (HSV-TK) and ganciclovir (GCV). Transduction of rhabdomyosarcoma cells using lentiviral vectors allowed efficient introduction of HSV-TK gene. In this study we proved high susceptibility of modified cells to ganciclovir resulting in eradication of cancer cells both in vitro and in vivo. Our data revealed strong gap junctional intercellular communication in examined cell line responsible for elimination of unmodified cells by bystander effect, even if HSV-TK-expressing cells comprise only 20% of cultured cells. Moreover, investigated approach is also efficient in vivo, where complete remission of tumors upon only 14 days of systemic administration of GCV can be observed. Obtained results suggest that HSV-TK suicide gene therapy is very promising concept in future clinical studies concerning rhabdomyosarcoma.