Author Summary Spatial smoothing is a preprocessing tool commonly applied to reduce the amount of noise in functional magnetic resonance imaging (fMRI) data. However, smoothing is known to affect the outcomes of functional brain network analysis at the level of individual subjects in undesired ways. Here, we investigate how spatial smoothing affects the observed differences in brain network structure between subject groups. Using fMRI data from two clinical populations and healthy controls, we show that the between-group differences in network structure depend on the amount of spatial smoothing applied during preprocessing in a nontrivial way. The optimal level of spatial smoothing is difficult to define and probably depends on a set of analysis parameters. Therefore, we recommend applying spatial smoothing only after careful consideration. Abstract Brain connectivity with functional magnetic resonance imaging (fMRI) is a popular approach for detecting differences between healthy and clinical populations. Before creating a functional brain network, the fMRI time series must undergo several preprocessing steps to control for artifacts and to improve data quality. However, preprocessing may affect the results in an undesirable way. Spatial smoothing, for example, is known to alter functional network structure. Yet, its effects on group-level network differences remain unknown. Here, we investigate the effects of spatial smoothing on the difference between patients and controls for two clinical conditions: autism spectrum disorder and bipolar disorder, considering fMRI data smoothed with Gaussian kernels (0–32 mm). We find that smoothing affects network differences between groups. For weighted networks, incrementing the smoothing kernel makes networks more different. For thresholded networks, larger smoothing kernels lead to more similar networks, although this depends on the network density. Smoothing also alters the effect sizes of the individual link differences. This is independent of the region of interest (ROI) size, but varies with link length. The effects of spatial smoothing are diverse, nontrivial, and difficult to predict. This has important consequences: The choice of smoothing kernel affects the observed network differences.