This study aimed to investigate the role of palmitic acid (PA) in modulating the neutrophil response during Trichomonas vaginalis infection. A human neutrophil-like cell model (nHL-60) was established by differentiating the human promyelocytic leukemia cell line HL-60 with 1.3% dimethyl sulfoxide. The expressions of the genes IL-8, NF-κB, MyD88, and TLR4 and the production of reactive oxygen species (ROS) of differentiated cells were compared, using end-point RT-PCR and nitroblue tetrazolium reduction, in the presence or absence of 1 mM PA and 100 μM of docosahexaenoic acid. Additionally, oxidative burst activity was also evaluated in response to T. vaginalis stimulation in the presence of either PA or docosahexaenoic acid. nHL-60 cells showed increased expression of key mediators of the TLR4 signaling pathway, and enhanced ROS production upon phorbol 12-myristate 13-acetate stimulation. Peripheral blood neutrophils exposed to T. vaginalis in the presence of PA exhibited higher ROS production than neutrophils stimulated by the parasite only. These findings indicate that PA enhances neutrophil activation to a secondary stimulus with T. vaginalis. They provide new insights into the role of metabolic factors in host-pathogen interactions.