Background <p>The cattle tick <i>Rhipicephalus microplus</i> is a major ectoparasite affecting livestock worldwide, and its increasing resistance to synthetic acaricides has intensified the search for plant-derived alternatives. Although the hydroalcoholic seed extract of <i>Randia aculeata</i> has demonstrated acaricidal activity, its molecular effects in ticks remain poorly understood. This study evaluated the acaricidal activity of the extract and the underlying molecular mechanism against <i>R. microplus</i> larvae.</p> Methods <p>The acaricidal activity was evaluated using the larval packet test, and the underlying mechanism was investigated through transcriptional analysis of acetylcholinesterase 2 (AChE2) and glutathione S-transferase (GST) genes. LC₅₀ and LC₉₀ values were estimated by probit regression analysis. Independent larval batches were exposed to the LC₅₀ concentration for 24&#xa0;h, and relative gene expression was quantified by quantitative real-time PCR.</p> Results <p>The extract demonstrated acaricidal activity, with 50% and 90% lethal concentration values of 2.84&#xa0;mg/mL and 5.67&#xa0;mg/mL, respectively. Exposure to the LC₅₀ concentration significantly reduced the expression of AChE2 (0.41   0.08-fold, <i>P</i> = 0.003) and GST (0.48   0.07-fold, <i>P</i> = 0.007) compared with controls.</p> Conclusions <p>The hydroalcoholic extract of <i>R. aculeata</i> exhibited acaricidal activity and was associated with transcriptional suppression of genes involved in neurotransmission and detoxification. These findings suggest that the extract may affect pathways related to cholinergic regulation and xenobiotic response. However, because the susceptibility status of the evaluated field-derived population was not determined, the observed molecular responses should be interpreted with caution. Further studies using well-characterized susceptible and resistant tick populations are needed to clarify the mechanisms underlying the biological activity of the extract.</p>