Excessive manganese (Mn) exposure may adversely affect the central nervous system, and cause an extrapyramidal disorder known as manganism. The glutamine (Gln)/glutamate (Glu)–γ-aminobutyric acid (GABA) cycle and thyroid hormone system may be involved in Mn-induced neurotoxicity. However, the effect of Mn on the Gln/Glu–GABA cycle in the serum has not been reported. Herein, the present study aimed to investigate the effects of sub-acute Mn exposure on the Gln/Glu–GABA cycle and thyroid hormones levels in the serum of rats, as well as their relationship. The results showed that sub-acute Mn exposure increased serum Mn levels with a correlation coefficient of 0.733. Furthermore, interruption of the Glu/Gln–GABA cycle in serum was found in Mn-exposed rats, as well as thyroid hormone disorder in the serum via increasing serum Glu levels, and decreasing serum Gln, GABA, triiodothyronine (T3) and thyroxine (T4) levels. Additionally, results of partial correlation showed that there was a close relationship between serum Mn levels and the detected indicators accompanied with a positive association between GABA and T3 levels, as well as Gln and T4 levels in the serum of Mn-exposed rats. Unexpectedly, there was no significant correlation between serum Glu and the serum T3 and T4 levels. In conclusion, the results demonstrated that both the Glu/Gln–GABA cycle and thyroid hormone system in the serum may play a potential role in Mn-induced neurotoxicity in rats. Thyroid hormone levels, T3 and T4, have a closer relationship with GABA and Gln levels, respectively, in the serum of rats.