Objective: The purpose of this study was to investigate the effects of a spinal thrust manipulation directed to the upper cervical segments (atlanto-occipital joint) on active mouth opening and pressure pain sensitivity in a trigeminal nerve innervated region (sphenoid bone) in women with mechanical neck pain. Methods: Thirty-seven women, ages 21 to 50 years old (mean age, 35 +/- 8 years) with mechanical neck pain were recruited for this study. Participants were randomly assigned into 1 of 2 groups as follows: an experimental group that received a spinal manipulation of the atlanto-occipital joint and a control group that received a manual contact placebo intervention. Outcomes collected were assessed pretreatment and 5 minutes posttreatment by an assessor blinded to the treatment allocation and included active mouth opening and pressure pain thresholds (PPTs) over both sides of the sphenoid bone. A 2-way repeated measures analysis of variance (ANOVA) with time (pre-post) as the within subjects variable and group (control, experimental) as the between subjects variable was used to examine the effects of the intervention. The hypothesis of interest was group-time interaction. Results: The ANOVA showed a significant effect for time (F = 23.1; P < .001) and an interaction between group and time (F = 37.7; P < .001) for active mouth opening as follows: the experimental group showed a greater improvement when compared to the control group. A large positive within-group effect size (d > 1.5) for the experimental group, whereas a negative medium within-group effect size (d = -0.5) for the control group were identified. The ANOVA showed a significant interaction between group and time (F = 14.4; P < .001) for PPT levels at the sphenoid bone as follows: the experimental group showed a greater improvement when compared to the control group. A medium positive within-group effect size (d = -0.5) for the experimental group, whereas a negative medium within-group effect size (d = -0.5) for the control group was found. Conclusions: Our findings suggest that the application of an atlantoaxial joint thrust manipulation resulted in an increase in active mouth opening and PPT over a trigeminal nerve distribution area (sphenoid bone) in women with mechanical neck pain.