Aims: Statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Methods and Results: Urea, K 2HPO4, chitin and yeast extract were identified as significant components influencing chitinase production by Paenibacillus sp. D1 using Plackett-Burman method. Response surface methodology (central composite design) was applied for further optimization. The concentrations of medium components for improved chitinase production were as follows (g l-1): urea, 0·33; K2HPO4, 1·17; MgSO4, 0·3; yeast extract, 0·65 and chitin, 3·75. This statistical optimization approach led to the production of 93·2 ± 0·58 U ml-1 of chitinase. Conclusions: The important factors controlling the production of chitinase by Paenibacillus sp. D1 were identified as urea, K2HPO4, chitin and yeast extract. Statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 2·56-fold increase in chitinase production. Significance and Impact of the Study: The present investigation provides a report on statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Paenibacillus species are gram-positive, spore-forming bacteria with several PGPR and biocontrol potentials. However, only few reports concerning mycolytic enzyme production especially chitinases are available. Chitinase produced by Paenibacillus sp. D1 represents new source for biotechnological and agricultural use. © 2009 No claims to Italian government works. © 2009 The Society for Applied Microbiology.