Maternal hypothyroidism has adverse effects on neural development in the offspring. The present study aimed to investigate whether maternal subclinical hypothyroidism impairs spatial learning in the offspring, as well as the efficacy and optimal time of levothyroxine (L-T4) treatment in pregnancy. Female adult Wistar rats were randomly divided into six groups (n = 10 per group): control, hypothyroid (H), subclinical hypothyroid (SCH) and SCH treated with L-T4, starting from the tenth, thirteenth and seventeenth gestational day (GD10, GD13 and GD17), respectively, to restore normal thyroid hormone levels. Spatial learning was assessed on progenies by a water maze test, a field excitatory postsynaptic potential (fEPSP) recording, and an long-term potentiation induction assay. Protein levels of early growth response protein 1 (Egr1), activity-regulated cytoskeleton-associated protein (Arc), Ras-proximate-1 (Rap1), p-extracellular signal-regulated kinase (p-ERK) and brain-derived neurotrophic factor (BDNF) were determined by western blotting. Progenies from the SCH and H groups demonstrated significantly longer mean latency in the water maze test and a lower amplification percentage of the amplitude and slope of the fEPSPs compared to offspring of the control group. L-T4 treatment for the GD10 and GD13 groups significantly shortened mean latency and increased the amplification percentage of the amplitude and slope of the fEPSPs of the progeny of rats with subclinical hypothyroidism. However, L-T4 treatment for the GD17 group showed only minimal effects on spatial learning in the offspring. Progenies of SCH and H groups had lower levels of Egr1, Arc, p-ERK and BDNF but higher levels of Rap1 compared to those of the controls. L-T4 treatment ameliorated these protein expression changes in the progeny of rats with subclinical hypothyroidism. Maternal subclinical hypothyroidism impaired spatial learning in the offspring; L-T4 treatment in early pregnancy recovered this adverse effect, and the optimal time of treatment should start from early pregnancy (GD10 and GD13).