Trace metals always act as cofactors or coenzymes in many cellular processes. Deficiency or excess of some metals will affect the fermentation of lignocellulosic hydrolysate. In order to make sure the deficient or excessive states of metals in culture medium, metal contents analysis was conducted in Pichia stipitis ATCC 58784 cells, synthetic medium, and diluted acid hydrolysate of rice straw. The results showed that Cu, Ni, and Co were deficient, and Al was a little excessive. So the influences of Cu(2+), Al(3+), Ni(2+), and Co(2+) additions on the growth and ethanol production of ATCC 58784 were further researched. Low concentration additions of Cu(2+) and Al(3+) (<0.24 mM and <0.23 mM, respectively) improved biomass growth of ATCC 58784 by 34 and 13%, respectively; however, higher concentrations decreased biomass growth. On the other hand, addition of Cu(2+) (0.39 mM) did not affect volumetric ethanol production significantly (P = 0.05) and addition of Al(3+) (0.38 mM) showed no influence on volumetric ethanol production (P = 0.68). Addition of 0.074 mM Co(2+) inhibited biomass growth of ATCC 58784 by 13% and volumetric ethanol production by 10%. The biomass growth and volumetric ethanol production of ATCC 58784 was arrested by the addition of 0.33 mM of Ni(2+) by 53 and 65%, respectively.