The current research was designed to assess possible differences in the emotional content of pleasant and unpleasant face emoji using acoustically evoked eyeblink startle reflex response. Stimuli were selected from Emojipedia Webpage. First, we assessed these stimuli with a previous independent sample of 190 undergraduate students (46 males and 144 females) mean age of 21.43 years (SD 3.89). A principal axis method was performed using the 30 selected emoji faces, extracting two factors (15 pleasant and 15 unpleasant emoji). Second, we measured the acoustic startle reflex modulation in 53 young adult women [mean age 22.13 years (SD 4.3)] during the viewing of each of the 30 emoji emotional faces in the context of the theory of motivation and emotion proposed by Lang (1995), but considering only the valence dimension. We expected to find higher acoustically evoked startle responses when viewing unpleasant emoji and lower responses for pleasant ones, similarly to the results obtained in the studies using human faces as emotional stimulus. An ANOVA was conducted to compare acoustic startle responses associated with pleasant and unpleasant emoji. Results yielded main effects for picture valence (λ = 0.80, F(1, 50) = 12.80, p = .001, η2 = 0.20). Post-hoc t test analysis indicated significant differences in the startle response between unpleasant (50.95 ± 1.75) and pleasant (49.14 ± 2.49) emoji (t (52) = 3.59, p = .001), with a Cohen's d = 0.495. Viewing affective facial emoji expressions modulates the acoustic startle reflex response according to their emotional content.