This study was conducted to investigate the effect of in ovo betaine (IOB) and thermal manipulation during incubation on growth performance, and some immune parameters of broilers under cyclic heat stress (CHS). Eggs were incubated under control (CL) and heat acclimation (HA) conditions. Betaine was injected into chicken embryos at day 11 of incubation (E11). Chicks were raised under standard management conditions until 21 d of age. From 21 to 42 d of age, half of the birds from each incubation treatment (IT) were exposed to CHS. The others were kept at 24°C (OPT). Betain and HA did not affect embryonic mortalities, hatchability, chick weight, and relative weights of digestive organs of chicks. Thymus and spleen weights of chicks increased with betain. The serum IgG was higher in HA + IOB-treated chicks. Blood cholesterol was not influenced by treatments. At 7 and 21 d, the body weights (BW) of chicks from IOB had heavier. At 28 and 35 d, there were significant interaction between IT × rearing temperature (RT) and IOB × RT on BW. Betain increased feed consumption (FC) and improved feed conversion (FCR) from 0 to 21 d. At 28 d, IT × RT interaction on FC was significant. At 35 d, there were significant interactions among IT × IOB × RT on FC and FCR. Betain slightly improved FCR from 0 to 42 d. These results indicated that HA and IOB positively affected thymus weight and IgG level of day-old chicks and enhanced broiler performance of birds under CHS.