Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection (LRTI) in children from infancy up to early childhood. Recently, we demonstrated that RSV infection alters cellular small non-coding RNA (sncRNA) expression, most notably the tRNA-derived RNA fragments (tRFs). However, the functions of the tRFs in virus host interaction are largely unknown. Herein, we examined the role of three RSV-induced tRFs derived from the 5-end of mature tRNAs decoding GIyCCC, LysCTT and CysGCA (named tRF5-GIyCCC, tRF5-LysCTT and tRF5-CysGCA, respectively) in controlling RSV replication. We found that tRF5-GIyCCC and tRF5-LysCTT, but not tRF5-CysGCA, promote RSV replication, demonstrating the functional specificity of tRFs. The associated molecular mechanisms underlying the functions of tRF5-GIyCCC and tRF5LysCTT were also investigated. Regulating the expression and/or activity of these tRFs may provide new insights into preventive and therapeutic strategies for RSV infection. The study also accumulated data for future development of a tRF targeting algorithm.