Background <p>Temperate phages play a central role in the evolution and pathogenicity of <i>Staphylococcus aureus</i>. Sa3int phages provide highly human-specific virulence factors that promote immune evasion and survival within the host. The reversible excision of these phages which occurs without phage production and bacterial lysis allows the simultaneous expression of phage virulence genes and the <i>hlb</i> gene where they usually integrate. However, the regulatory mechanisms that control phage assembly and the cross-talk with host factors remain poorly understood.</p> Methods and results <p>We analyzed the regulatory mechanism controlling late gene transcription of Sa3int phage Φ13. We identified a functional promoter, P_23, located upstream of the late phage genes that control DNA processing and packaging, capsid assembly, bacterial lysis and immune evasion. <i>SAOUHSC_02200</i>, the gene located upstream of P₂₃, encodes for a late transcriptional regulator (Ltr). Mutating the P₂₃ TATA-box or the <i>ltr</i> gene abolished P₂₃ activity and formation of mature intact phage particles, thus confirming the role of Ltr in regulating P₂₃ activity. Four direct repeats upstream of the P₂₃ transcriptional start site were identified as potential Ltr binding sites. RT-qPCR analysis confirmed that Ltr-dependent P₂₃ activation is essential for the expression of late genes and the subsequent Φ13 propagation. Furthermore, comparative analysis of P₂₃ activity and <i>ltr</i> expression in different host strain backgrounds revealed strain-specific differences that appear to depend on the alternative sigma factor SigB and its downstream effector SpoVG.</p> Conclusions <p>Ltr controls the expression of late phage genes, thereby regulating phage assembly and lysis. This process is modulated by SpoVG activity.</p>