Phosphine, which is released continuously from sediment, can affect the eco-physiological strategies and molecular responses of phytoplankton. To examine the effects of phosphine on phosphorus uptake and utilization in Thalassiosira pseudonana, we examined the transcriptional level of the phosphate transporter gene (TpPHO) and the activity of alkaline phosphatase (AKP) in relation to supplement of various concentrations of phosphine. TpPHO expression was markedly promoted by phosphine in both the phosphate-deficient and phosphate-4 µM culture. However, high phosphine concentrations can inhibit TpPHO transcription in the declining growth phase. AKP activity was also higher in the phosphine treatment groups than that of the control. It increased with increasing phosphine concentration in the range of 0 to 0.056 µM but was inhibited by higher levels of phosphine. These responses revealed that phosphine can affect phosphate uptake and utilization in T. pseudonana. This result was consistent with the effect of phosphine on algal growth, while TpPHO expression and AKP were even more sensitive to phosphine than algal growth. This work provides a basic understanding for further research about how phosphine affects phytoplankton.