Aims To clarify the effects of Phellinus baumii ethanol extract (PBE) on Brucella abortus pathogenesis in phagocytes focusing on the phagocytic and intracellular trafficking pathway. Methods and Results The effects of PBE on Br. abortus infection in macrophages were evaluated through an adherence and infection assays and an analysis of LAMP-1 staining. The phosphorylation of ERK1/2 and the F-actin polymerization associated with PBE during Br. abortus uptake were detected by immunoblotting and FACS, respectively. The survival of Br. abortus in pure culture was remarkably reduced by PBE in a dose-dependent manner. PBE-treated cells showed significantly decreased uptake, intracellular replication and adherence of Br. abortus. The declines of ERK1/2 phosphorylation and F-actin polymerization following Br. abortus entry were apparent in PBE-treated cells compared with the control. Moreover, the co-localization of Br. abortus-containing phagosomes with LAMP-1 was elevated in PBE-treated cells compared with the control during intracellular trafficking. Conclusion Phellinus baumii ethanol extract may possess the modulatory effect on pathogenesis of Br. abortus through disrupting the phagocytic and intracellular trafficking pathway in phagocyte. Significance and Impact of the Study The potential modulation of PBE to Br. abortus pathogenesis could provide an alternative approach to control of brucellosis, contributing to attenuate Br. abortus manifestation in hosts.