Opuntia ficus-indica is a drought-tolerant cactus that is increasingly becoming popular due to its nutritional and economic value in climate vulnerable regions. Its large-scale production in subtropical humid regions is constrained by high microbial contamination during in vitro establishment and poor survival during acclimatization.. So, this study aimed to develop and validate a climate-adapted micropropagation and acclimatization protocol optimized for humid subtropical conditions, with specific emphasis on sterilization efficiency, cytokinin-auxin interactions, and ex vitro survival.. For these, surface sterilization, cytokinin-mediated shoot induction, auxin-induced rooting, and ex vitro acclimatization substrates were systematically optimized. The result recorded that sequential treatment with 70% ethanol and 5% NaOCl achieved the highest sterilization efficiency (95%) with minimal tissue damage. Optimal shoot proliferation (3.0 shoots explant-1, 90% response) was observed on MS medium with BAP + KIN (2.5 + 0.5 mg L-1). In contrast, robust rooting (5 roots shoot-1, 95% success rate) was recorded in 1.5 mg L-1 IBA on half-strength MS. A 1:1 soil-sand mixture gave the highest acclimatization rate (92%). Unlike previous protocols developed for arid zones, this study demonstrated the first climate-specific adaptation of O. ficus-indica micropropagation to a humid subtropical environment, addressing contamination challenges by ensuring high ex vitro survival. The protocol provides a scalable, cost-effective strategy for future sustainable cactus cultivation in drought-prone environments.