Reproduction and locomotion are essential features of animals that help to facilitate their interaction with the surrounding environment. Previous studies have produced inconsistent results on behavioral response to spaceflight by the model animal Caenorhabditis elegans (C. elegans) in liquid culture. Using standard agar-based nematode growth medium (NGM), we show here that both reproductive and locomotory capacities of C. elegans were not significantly changed by centrifuge-produced hypergravity or clinostat-simulated microgravity. To investigate the effect of actual spaceflight on C. elegans, a nematode test unit was specifically designed to maintain its normal growth on solid NGM slides and to allow automatic RNA fixation on board the Shenzhou-8 spaceflight. We did not detect alteration in either brood size of immediate progenies from postflight nematodes or locomotory behavior, including speed of locomotion, frequency of reversals, and rate of body bends of space-flown nematodes collected directly from nematode test units. Our results provide clear evidence that the nematode test unit is an appropriate apparatus for nematode growth on standard NGM and can be used for on-orbit analysis of C. elegans, including onboard RNA fixation for molecular analysis and real-time video acquisition for behavioral analysis, which are critical for further studies in unmanned spaceflight and outer space exploration.