This study was aimed to investigate the feasibility of using simulated microgravity to study the effects of microgravity on activity and sleep of Drosophila melanogaster. Simulated microgravity experiments performed using the random positioning machine (RPM) and male Drosophila melanogaster, and the activity and sleep of the flies were monitored using the DAM system (Trikinetics, USA). First, flies were exposed to simulated microgravity for a short time (3 days) with DAM system monitoring. Next, after being exposed to simulated microgravity for a long time (10, 20, and 30 days), flies were removed to be monitored for 3 consecutive days under control conditions. Then, the flies after short-term treatment (3 days) with greater impact were sampled to study the effects of simulated microgravity on the major circadian clock genes period (per), timeless (tim), clock (clk), cycle (cyc), cryptochrome (cry), and ddc, pale, trh genes encoding dopa decarboxylase, tyrosine hydroxylase, tryptophan hydroxylase, that involved in synthesis of neurotransmitter dopamine (DA) and 5-hydroxy-tryptamine (5-HT), and the contents of DA and 5-HT. The results showed that during short-term treatment, total activity accounts and activity accounts in wake period both increased, and total sleep and number of sleep episode both decreased at night; after short-term treatment, the relative expression levels of tim, clk, cyc and cry increased, and the relative expression levels of ddc, pale and trh increased; after long-term treatment, overall effect on activity and sleep was slight. This study suggests that it is feasible to study the effects of microgravity on activity and sleep of Drosophila melanogaster by simulated microgravity, and valuable reference can be provided for aerospace medical research.