γ-aminobutyric acid (GABA) is an amino acid used for mitigating the detrimental effects of heat stress in broilers. In addition, a growing body of literature suggests that the in ovo feeding of various nutrients can enhance the post-hatch thermotolerance of broilers. Therefore, we hypothesized that the supplementation of GABA during incubation might have positive effects in heat-stressed broilers. Chicks hatched from eggs were divided into three groups described as follows: chicks hatched from eggs incubated at normal temperature and then raised under thermoneutral temperature (CON); chicks hatched from eggs incubated at normal temperature but raised under cyclic heat stress (HS) (CON+HS); and chicks hatched from eggs injected with 60 mg of GABA dissolved in 0.6 mL of distilled water but raised under cyclic HS (G10+HS). The HS was applied between 28 and 31 days of age with ambient temperatures raised from 22 ± 1 °C to 33 ± 1 °C for 6 h daily. Compared to the CON group, average daily weight gain was significantly lower in the CON+HS but not in the G10+HS group. Feed intake was significantly decreased in both the CON+HS and G10+HS groups. Compared to the CON group, plasma corticosterone levels were significantly increased in the CON+HS group, but not the G10+HS group. Hepatic mRNA levels of the acetyl-CoA carboxylase gene (ACC) were significantly reduced in the G10+HS group compared to the CON group. In addition, positive Pearson correlation coefficients were found in mRNA levels between fatty acid synthase (FAS) and nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) (r = 0.55, p < 0.05), NOX1 and NOX4 (r = 0.65, p < 0.01), and catalase (CAT) and superoxide dismutase (SOD) (r = 0.62, p < 0.05). Taken together, the results suggest that this study can serve as a basis for future work focusing on the in ovo feeding of GABA as a technique to combat heat stress in broilers.