The preservation and development of Sipahutar pineapple is very important to do. One of the most effective ways is with tissue culture techniques. The aim of this research is to know the effect of 2,4-Diclorofenoxyacetic acid (2,4-D), kinetin, and interaction of both Plant Growth Regulator (PGR) to callus induction on pineapple (Ananas comosus L.) from Sipahutar Sumatera Utara Indonesia. The design of this study used a Factorial Completely Randomized Design, namely is 2.4-D with 3 doses of treatment (0, 1, 2 ppm) as the first and the second factor of kinetin with 3 doses (0, 0.5, 1 ppm). The observation process was conducted for 28 days and 35 days after induction. The parameters observed were the formation time of callus, callus color, callus biomass, and the height of callus pile. The results showed that 2,4-D, kinetin and interaction of 2,4-D and kinetin significantly influenced the time of callus formation, callus biomass, and significant effect on the height of callus pile. The time of callus formation is the fastest on day 8 after induction. The best of callus color is whitish green comes from 2,4-D 0 ppm and kinetin 1 ppm treatment. The highest biomass is 1.69 grams. The highest stack height of callus was 2.32 cm. The highest biomass, stack height, surface area of callus derived from the treatment of 2,4-D 0 ppm and kinetin 0 ppm.