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Background: Streptococcus pneumoniae is a leading pathogen worldwide and its pathogenesis remains unclear. Objectives: The study aimed to investigate the secretion of interleukin-8 (IL-8) and soluble intercellular adhesion molecule-1 (ICAM-1) from A549 pneumocytes stimulated by different S. pneumoniae strains and the mechanism of blood-derived S. pneumoniae (bd-SP) in invading the blood system. Methods: Twenty-three clinical strains of S. pneumoniae isolated in 2009 along with ATCC49619 were cultured and suspended in 2018. Then, A549 pneumocytes were resuspended after culture and collection, and inoculated into culture plates. Streptococcus pneumoniae suspensions were then inoculated. Normal saline was blank control. The plates were incubated for four and eight hours. Then, the suspensions were collected and centrifuged. The supernatant was analyzed for IL-8 and ICAM-1 by ELISA. Results: The concentrations of IL-8 were 180.6, 188.5, 223.4 ± 19.9, and 230.3 ± 38.6 pg/mL for blank control, ATCC49619, blood-derived S. pneumoniae (bd-SP) and sputum-derived S. pneumoniae (sd-SP) at four-hour stimulation, respectively, and 249.2, 275.7, 224.0 ± 27.8, and 242.3 ± 33.1 pg/mL at eight-hour stimulation. The concentrations of ICAM-1 were 14.8, 12.1, 19.9 ± 17.2, and 26.1 ± 28.6 ng/mL for blank control, ATCC49619, bd-SP, and sd-SP at 4 hours, respectively, and 32.6, 150.8, 69.4 ± 45.1, and 58.7 ± 30.1 ng/mL at 8 hours. Conclusions: Streptococcus pneumoniae could upregulate the secretion of IL-8 and ICAM-1 from A549 pneumocytes. No significant difference was found between bd-SP and sd-SP which was in contrast to what was found between clinical S. pneumoniae and ATCC49619. Host response may not be a vital factor for different S. pneumoniae infections.