AimNeuroimaging studies have highlighted gender differences in brain functions, but conclusions are not well established. Few studies paid attention to the influence of phase-encoding (PE) direction in echo-planar imaging on gender differences, which is a commonly used technique in functional magnetic resonance imaging (fMRI). A disadvantage of echo-planar images is the geometrical distortion and signal loss due to large susceptibility effects along the PE direction. The present research aimed to clarify how PE direction can affect the outcome of a specific research on gender differences. MethodsWe collected resting-state fMRI using anterior to posterior (AP) and posterior to anterior (PA) directions from 113 healthy participants. We calculated several commonly used indices for spontaneous brain activity including amplitude of low frequency fluctuations (ALFF), fractional ALFF (fALFF), regional homogeneity (ReHo), degree centrality (DC), and functional connectivity (FC) of posterior cingulate cortex for each session, and performed three group comparisons: (i) AP versus PA; (ii) male versus female; (iii) interaction between gender and PE direction. ResultsThe estimated indices differed substantially between the two PE directions, and the regions that exhibited differences were roughly similar for all the indices. In addition, we found that multiple brain regions showed gender differences in these estimated indices. Further, we observed an interaction effect between gender and PE direction in the bilateral middle frontal gyrus, right precentral gyrus, right postcentral gyrus, right lingual gyrus, and bilateral cerebellum posterior lobe. ConclusionThese apparent findings revealed that PE direction can partially influence gender differences in spontaneous brain activity of resting-state fMRI. Therefore, future studies should document the adopted PE direction and appropriate selection of PE direction will be important in future resting-state fMRI studies.