In this study, the effects of in ovo feeding of γ-aminobutyric acid (GABA) and embryonic thermal manipulation (TM) on plasma biochemical parameters, organ weights, and hepatic gene expression in broilers exposed to cyclic heat stress (32 ± 1°C for 8 days) (HS) were investigated. A total of 175 chicks were assigned to five treatments: chicks hatched from control eggs (CON); chicks hatched from control eggs but exposed to HS (CON+HS); chicks hatched from eggs injected at 17.5 days of incubation with 0.6mL of 10% GABA and exposed to HS (G10+HS); chicks hatched from thermally manipulated eggs (39.6°C, 6h/d from embryonic days 10 to 18) and exposed to HS (TM+HS); chicks hatched from eggs that received both previous treatments during incubation and exposed to HS (G10+TM+HS). Results revealed that on day 36 post-hatch, hepatic NADPH oxidase 1 (P = 0.034) and 4 (P = 0.021) genes were downregulated in the TM+HS and G10+TM+HS compared to the CON+HS group. In addition, while acetyl-CoA carboxylase gene expression was reduced (P = 0.002) in the G10+TM group, gene expression of extracellular fatty acid-binding protein and peroxisome proliferator-activated receptor-γ was lower (P = 0.045) in the TM+HS group than in the CON+HS group. HS led to higher gene expression of heat shock protein 70 (HSP70) and 90 (HSP90) (P = 0.005, and P = 0.022). On the other hand, the TM+HS group exhibited lower expression of both HSP70 (P = 0.031) and HSP90 (P = 0.043) whereas the G10+TM+HS group had a reduced (P = 0.016) HSP90 expression compared to the CON+HS. MANOVA on different gene sets highlighted an overall lower (P = 0.034) oxidative stress and lower (P = 0.035) heat shock protein expression in the G10+TM+HS group compared to the CON+HS group. Taken together, the current results suggest that the combination of in ovo feeding of GABA with TM can modulate HSPs and antioxidant-related gene expression in heat-stressed broilers.