Bipolar disorder has been associated with disturbances in circadian rhythms. Lithium is frequently used in the long-term treatment of bipolar disorder, and has been shown to prolong such rhythms in animals and humans. To examine whether lithium affects the expression of genes regulating the circadian clock, cultured NIH-3T3 cells were synchronized by serum-shocking, and the relative expression of the clock genes Period1 (Per1), Period2 (Per2), Period3 (Per3), Cryptochrome1 (Cry1), Cryptochrome2 (Cry2), Brain and muscle aryl hydrocarbon nuclear translocator-like 1 (Bmal1), Circadian locomotor output cycles kaput (Clock), Rev-Erb-α (Nr1d1), RAR-related orphan receptor α (Ror-α), Glycogen synthase kinase-3β (Gsk-3β), Casein kinase 1-ε (CK1-ε; Csnk1ε), E4 binding protein 4 (E4BP4; Nfil-3) and albumin D-binding protein (Dbp) was examined for three consecutive days in the presence of lithium (20 mM) or vehicle (20 mM NaCl). We found that lithium significantly increased the expression of Per2 and Cry1, whereas Per3, Cry2, Bmal1, E4BP4 and Rev-Erb-α expression was reduced. We also found that lithium prolonged the period of Per2. Taken together, these effects on clock gene expression may be relevant for the effects of lithium on biological rhythms and could also give new leads to further explore its mood-stabilizing actions in the treatment of bipolar disorder.