African swine fever virus (ASFV) is a highly pathogenic agent that poses a severe threat to the global swine industry. The biological function of A859L, an ASFV-encoded RNA helicase, remains largely elusive. Here, we show that A859L is highly conserved across diverse ASFV isolates and belongs to the SF2 superfamily of RNA helicases, possessing characteristic Walker A (GKT) and Walker B (DECH) motifs. Transcriptomic profiling at multiple time points post-infection further revealed that A859L is a late-expressed gene. Functional assays demonstrated that siRNA-mediated knockdown of A859L significantly inhibited progeny virus production in both susceptible macrophages (PAMs and BMDMs) and WSL cells. This knockdown also markedly reduced the expression of viral proteins p30 and p72, as well as the intracellular mRNA levels of other viral helicase genes (QP509L, Q706L, D1133L, and B962L), underscoring the vital role of A859L in the viral life cycle. Conversely, A859L overexpression enhanced viral propagation. Notably, pharmacological inhibition of viral helicase activity effectively suppressed ASFV replication. Collectively, our data establish A859L as a critical determinant of ASFV replication and a promising candidate for antiviral development.