Paphiopedilum spp. is one of the most commercially popular orchids because of its variety of shapes, sizes, and colors. However, it is at risk for extinction because of its exploitation. Regeneration of orchid plants using internode segments is extremely difficult. In this study, young P. callosum plants (1.5 cm) were exposed to eight dark–light cycles (14 days of dark and 1 day of light) for stem elongation to increase the number of nodes to obtain internode tissues. After 75 days of culture, the highest callogenesis (31.25%) was achieved when internode tissue was cultured on liquid Schenk and Hildebrandt (SH) medium containing 30 g·L−1 sucrose, 1.0 mg·L−1 Thidiazuron (TDZ), 1.0 mg·L−1 2,4-Dichlorophenoxyacetic acid (2,4-D), and cotton wool as the support matrix. The optimal media for induction of protocorm-like bodies (PLBs) were the same compositions as previously mentioned and were supplemented with 9 g·L−1 Bacto agar as the gelling agent. PLB clumps (5–6 PLBs/clump) produced the best shoots on medium containing 0.5 mg·L−1 α-Naphthaleneacetic acid (NAA) and 0.3 mg·L−1 TDZ. Among the organic substances tested, 200 g·L−1 potato homogenate (PH) added to Hyponex N016 medium supplemented with 1.0 mg·L−1 NAA, 30 g·L−1 sucrose, 170 mg·L−1 NaH2PO4, 1.0 g·L−1 peptone, and 9 g·L−1 Bacto agar resulted in the best rooting. The rooted plantlets with four to five leaves were acclimatized and had a 100% survival rate. The method presented in this research provides a strategy for the development of highly effective propagation of Paphiopedilum species using ex vitro explants for both conservation and horticultural purposes.