Before plants can be effectively utilised as a component of enclosed life-support systems for space exploration, it is important to understand the molecular mechanisms by which they develop in microgravity. Using the Biological Research in Canisters (BRIC) hardware on board the second to the last flight of the Space Shuttle Discovery (STS-131 mission), we studied how microgravity impacts root growth in Arabidopsis thaliana. Ground-based studies showed that the actin cytoskeleton negatively regulates root gravity responses on Earth, leading us to hypothesise that actin might also be an important modulator of root growth behaviour in space. We investigated how microgravity impacted root growth of wild type (ecotype Columbia) and a mutant (act2-3) disrupted in a root-expressed vegetative actin isoform (ACTIN2). Roots of etiolated wild-type and act2-3 seedlings grown in space skewed vigorously toward the left, which was unexpected given the reduced directional cue provided by gravity. The left-handed directional root growth in space was more pronounced in act2-3 mutants than wild type. To quantify differences in root orientation of these two genotypes in space, we developed an algorithm where single root images were converted into binary images using computational edge detection methods. Binary images were processed with Fast Fourier Transformation (FFT), and histogram and entropy were used to determine spectral distribution, such that high entropy values corresponded to roots that deviated more strongly from linear orientation whereas low entropy values represented straight roots. We found that act2-3 roots had a statistically stronger skewing/coiling response than wild-type roots, but such differences were not apparent on Earth. Ultrastructural studies revealed that newly developed cell walls of space-grown act2-3 roots were more severely disrupted compared to space-grown wild type, and ground control wild-type and act2-3 roots. Collectively, our results provide evidence that, like root gravity responses on Earth, endogenous directional growth patterns of roots in microgravity are suppressed by the actin cytoskeleton. Modulation of root growth in space by actin could be facilitated in part through its impact on cell wall architecture.